bluuebunny/crossref_metadata_2025_split huggingface dataset:
The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase
Authors: Pisa René , Stein Torsten , Eichler Robert , Gross Roland , Simon Jörg 
Published: March 2003

SummaryThe cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem‐binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenesΔnrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix‐assisted laser‐desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild‐type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild‐type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.
Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion
Authors: Brayton Kelly A. , Palmer Guy H. , Lundgren Anna , Yi Jooyoung , Barbet Anthony F. 
Published: March 2003

SummaryThe rickettsial pathogen Anaplasma marginale establishes lifelong persistent
infection in the mammalian reservoir host, during which time immune escape variants
continually arise in part because of variation in the expressed copy of the immunodominant
outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale
genome generates sufficient variants to allow long‐term persistence in an immunocompetent
reservoir host. The recombination of whole pseudogenes into the single msp2
expression site has been previously identified as one method of generating variants,
but is inadequate to generate the number of variants required for persistent infection.
In the present study, we demonstrate that recombination of a whole pseudogene is
followed by a second level of variation in which small segments of pseudogenes recombine
into the expression site by gene conversion. Evidence for four short sequential changes
in the hypervariable region of msp2 coupled with the identification of nine
pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.
ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae
Authors: Li Caiyi C. , Merrell D. Scott , Camilli Andrew , Kaper James B. 
Published: March 2003

SummaryIn pathogenic Vibrio cholerae, the transmembrane DNA‐binding protein ToxR

co‐ordinates the expression of over 20 genes, including those encoding important

virulence factors such as cholera toxin and the toxin‐co‐regulated pilus. The outer

membrane protein OmpT is the only member of the ToxR regulon known to be repressed

by ToxR. In this study, we examined the environmental conditions that regulate OmpT

expression and demonstrated that ompT transcription is upregulated 14‐fold

when the bacteria enter late log phase from early log phase. Deletion of the crp

gene completely abolishes OmpT expression. Comparison of ompT transcription

levels in the isogenic crp−, toxR−

and crp−toxR− mutants

revealed that (i) in the absence of ToxR, constitutive high‐level ompT transcription

is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP‐dependent

ompT activation, but also abolishes the CRP‐independent, basal level ompT

transcription; thus, the mechanism by which ToxR represses ompT transcription

involves both antiactivation and direct repression; (iii) both CRP and ToxR are required

for the regulation of OmpT expression by growth phase. To provide further insights

into the molecular mecha‐nism of CRP‐dependent activation of ompT transcription,

we demonstrated that CRP‐dependent activation requires a CRP binding site centred

at −310 of the ompT promoter, without which the interaction of

CRP with other CRP binding site(s) more proximal to the promoter results in repression.

Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase

(RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from −310

of the ompT promoter via DNA looping.
RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli
Authors: Lee Kangseok , Bernstein Jonathan A. , Cohen Stanley N. 
Published: March 2003

SummaryThe Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have

sequence similarity and broadly similar sequence specificity. Whereas the absence

of Rne normally is lethal, we show here that E. coli bacteria that lack the

rne gene can be made viable by overexpression of Rng. Rng‐complemented cells

accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase

P (i.e. M1 RNA), indicating that normal processing of these Rne‐cleaved RNAs was

not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from

precursors by RNase G cleavage in vitro. Using DNA microarrays containing

4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose

steady‐state level was affected by Rne, Rng or the N‐terminal catalytic domain of

RNase E. Most transcript species affected by RNase E deficiency were also elevated

in an rne deletion mutant complemented by Rng. However, approximately 100

mRNAs that accumulated in Rne‐deficient cells were decreased by rng‐complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy‐generating pathways or in the synthesis or degradation of macromolecules.
Regulation of autolysins in teichuronic acid‐containingBacillus subtiliscells
Authors: Calamita Heather G. , Doyle R. J. 
Published: March 2003

SummaryBacillus subtiliscells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA). The turnover rates of TA‐containing and TUA‐containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar. In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)–dextran to probe the wall pH, that a low pH exists in the wall matrix. A second probe, cationized ferritin (CF), was used to observe cell surface protonation. Suspensions ofB. subtiliscells containing either TA or TUA were aggregated with CF only after the addition of a protonmotive force‐dissipating agent. RespiringB. subtilisTUA‐containing cells labelled with FITC–dextran exhibited little fluorescence. Conversely, fluorescence intensities exhibited by cells de‐energized with nitrogen gas were significantly greater. The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA‐containing and TUA‐containingB. subtiliscells. Both TUA‐containing and TA‐containing cells were lysed only after the addition of sodium azide. These data suggest that during normal growth the wall of TUA‐containingB.subtilis cells is protonated, and protonmotive force influences autolytic regulation in both TUA‐containing and TA‐containing B. subtilis cells.
Binding site requirements of the virulence gene regulator AphB: differential affinities for the Vibrio cholerae classical and El Tor tcpPH promoters
Authors: Kovacikova Gabriela , Skorupski Karen 
Published: March 2003

SummaryThe differential expression of virulence genes be‐tween the two disease‐causing biotypes of Vibrio cholerae, classical and El Tor, is primarily due to a single basepair change in the tcpPH promoter, which strongly influences the ability of the LysR regulator AphB to activate transcription in response to environmental conditions. We show here that this single basepair change influences virulence gene expression by dramatically altering the affinity of AphB for its recognition site in the tcpPH promoter. AphB binds greater than 10‐fold more efficiently to a wild‐type classical tcpPH promoter fragment with an A at −65 relative to a wild‐type El Tor fragment that has a G at this position. As this single basepair change is located within the left arm of the LysR recognition motif (5′‐TGCAA‐N7‐TTGCA), which extends from −69 to −53, a systematic mutagenesis of the other positions within this site was carried out to assess their influence on AphB binding in vitro and transcriptional activation in vivo. This analysis revealed that the left and right arms of the interrupted dyad display a high degree of symmetry with respect to their role in AphB binding. The right promoter proximal arm also plays a role in transcriptional activation that is distinct from its role in AphB binding. A second AphB binding site (5′‐TGCAA‐N7‐TGTCA) was identified upstream of the aphB gene itself, which extends from +17 to +33 relative to the start of transcription and functions in autorepression. Although the sequences of the AphB binding sites at the tcpPH and aphB promoters are highly conserved, important differences exist in the way that AphB functions at each of these sites.
The cyanobacterial phytochrome Cph2 inhibits phototaxis towards blue light
Authors: Wilde Annegret , Fiedler Brita , Börner Thomas 
Published: March 2003

SummaryWe analysed the effects of inactivation of the phytochrome genes cph1 and
cph2 on the phototactic migration of the cyanobacterium Synechocystis
sp. PCC 6803 under different light qualities. Motility towards white, green, red
and far red light was not altered in several independently raised cph1, cph2
and cph1/cph2 double mutants. Blue light (λ = 400–500
nm) did not induce motility in wild‐type and cph1 mutant cells, whereas cph2
and cph1/cph2 double mutants moved towards the blue light. Inhibition
of the photosynthetic electron transport by DCMU influenced the motility of cph2
mutants under blue light, but not under white, red and far red light. RNA blot hybridizations
did not indicate an altered transcript accumulation of the pilin‐encoding pilA1
gene under blue light. We propose that the Cph2 protein is part of a light‐stimulated
signal transduction chain inhibiting the movement of Synechocystis sp. PCC 6803 cells towards blue light.
Differential gene expression in Entamoeba histolytica isolated from amoebic liver abscess
Authors: Bruchhaus Iris , Roeder Thomas , Lotter Hannelore , Schwerdtfeger Maike , Tannich Egbert 
Published: March 2003

SummaryThe majority of human infections with the intestinal protozoan parasite Entamoeba histolytica remain asymptomatic. In a small proportion of infections, however, E. histolytica trophozoites penetrate the intestinal mucosa and disseminate to other organs, most commonly to the liver, where they induce abscess formation. It is believed that the ability of E. histolytica trophozoites to destroy host tissues and to survive within the liver is accomplished by a strong adaptive response, which requires the specific regulation of a number of amoeba proteins. Using differential display polymerase chain reaction (DD‐PCR), we compared RNA expression between E. histolytica trophozoites isolated from liver abscesses of infected gerbils and those grown under normal culture conditions. A total of 3000 cDNA‐derived amplicons were compared between the two groups of amoebae, which were calculated to represent about one‐third of all E. histolytica mRNA species (transcriptome). Among these, 55 were found to be specifically present or absent in abscess‐derived amoebae, of which 42 were successfully cloned and sequenced. Database searches and Northern blot analyses revealed that the 42 amplicons correspond to 29 independent E. histolytica genes, of which at least seven are specifically upregulated and five are downregulated in abscess‐derived amoebae. Specific expression of most of these genes was not simply the result of a heat shock response, which might be expected during abscess formation, as only five of the genes revealed an expression profile similar to that found in amoebae cultured under elevated temperatures. The two genes specifically downregulated in abscess‐derived amoebae encode members of a family of so far unknown proteins, which contain repetitive stretches of sequences that are rich in lysine and glutamic acid residues. In contrast, a diverse set of genes is specifically upregulated, encoding ribosomal proteins (S30, L37A), cyclophilin, ferredoxin 2 and GTP‐binding protein RAB7D, supporting the notion that liver abscess formation requires the regu‐lation and concerted action of a variety of amoeba proteins. These proteins are associated with stress response, signal transduction, regulation of transcription and vesicular trafficking. However, transcriptome analysis will not be sufficient to identify all proteins specifically upregulated during abscess formation, as at least an increase in the expression of actin was found to be regulated at the post‐transcriptional level.
A novel cyclic GMP‐dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle
Authors: Deng Wensheng , Baker David A. 
Published: March 2003

SummaryCyclic GMP‐dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. We report here the characterization of an unusual PKG from the human malaria parasite Plasmodium
falciparum (designated PfPKG). The 97.5 kDa protein contains some of the structural
features of mammalian PKGs but, uniquely, contains a third predicted cGMP binding
site and a degenerate fourth. Using both protein kinase activity assays and Western
blotting with native P. falciparum proteins, we demonstrate here that PfPKG
is expressed predominantly in the ring stage of the life cycle, suggesting a role
in the development of asexual blood stage parasites. An Escherichia coli‐derived
recombinant protein (PfPKG2, Met115‐Phe853) was purified and shown to have phosphotransferase activity in terms of both substrate phosphorylation and auto‐phosphorylation. This activity was stimulated at least fivefold by 1.0 μ M cyclic GMP, but was not stimulated by cAMP or by 8‐pCPT‐cGMP, which is a potent activator of mammalian PKGs. Several protein kinase inhibitors exhibited a range of inhibitory effects on PfPKG activity. Biochemical analysis therefore shows that PfPKG is distinct from mammalian PKGs with respect to both cyclic nucleotide analogue activation and inhibition profiles.
The ShcA protein is a molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein secretion system of Pseudomonas syringae
Authors: Dijk Karin van , Tam Vincent C. , Records Angela R. , Petnicki‐Ocwieja Tanja , Alfano James R. 
Published: March 2003

Summary Pseudomonas syringae uses a type III protein secretion system encoded by
the Hrp pathogenicity island (Pai) to translocate effector proteins into plant cells.
One of these effector proteins is HopPsyA. A small open reading frame (ORF), named
shcA, precedes the hopPsyA gene in the Hrp Pai of P. s. syringae
61. The predicted amino acid sequence of shcA shares general characteristics
with chaperones used in type III protein secretion systems of animal pathogens. A
functionally non‐polar deletion of shcA in P. s. syringae 61 resulted
in the loss of detectable HopPsyA in supernatant fractions, consistent with ShcA
acting as a chaperone for HopPsyA. Cosmid pHIR11 carries a functional set of type
III genes from P. s. syringae 61 and confers upon saprophytes the ability
to secrete HopPsyA in culture and to elicit a HopPsyA‐dependent hypersensitive response
(HR) on tobacco. P. fluorescens carrying a pHIR11 derivative lacking shcA
failed to secrete HopPsyA in culture, but maintained the ability to secrete another
type III‐secreted protein, HrpZ. This pHIR11 derivative was also greatly reduced
in its ability to elicit an HR, indicating that the ability to translocate HopPsyA
into plant cells was compromised. Using affinity chromatography, we showed that ShcA
binds directly to HopPsyA and that the ShcA binding site must reside within the first
166 amino acids of HopPsyA. Thus, ShcA represents the first demonstrated chaperone
used in a type III secretion system of a bacterial plant pathogen. We searched known
P. syringae type III‐related genes for neighbouring ORFs that shared the general characteristics of type III chaperones and identified five additional candidate type III chaperones. Therefore, it is likely that chaperones are as prevalent in bacterial plant pathogen type III systems as they are in their animal pathogenic counterparts.
Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data
Authors: Martin Robert G. , Rosner Judah L. 
Published: March 2003

SummaryMicroarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.
The autoregulatory role of EsaR, a quorum‐sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function
Authors: Minogue Timothy D. , Trebra Markus Wehland‐von , Bernhard Frank , Bodman Susanne B. von 
Published: March 2003

SummaryCapsular polysaccharide synthesis and virulence in the plant pathogenic bacterium Pantoea stewartii ssp. stewartii requires the quorum‐sensing regulatory proteins, EsaR and EsaI, and the diffusible inducer N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone. Prior mu‐tational studies suggested that EsaR might function as a repressor of quorum sensing in the control of capsular polysaccharide synthesis. Further, a lux box‐like palindromic sequence coinciding with the putative –10 element of the esaR promoter suggested a possible negative autoregulatory role for EsaR. This report presents genetic evidence that EsaR represses the esaR gene under inducer‐limiting conditions, and that addition of inducer promotes rapid, dose‐dependent derepression. DNA mobility‐shift assays and analyses by surface plasmon resonance refractometry show that EsaR binds target DNAs in a ligand‐free state, and that inducer alters the binding characteristics of EsaR. Physical measurements indicate that the EsaR protein binds N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone, in a 1:1 protein:ligand ratio, and that inducer binding enhances the thermal stability of the EsaR protein. These combined genetic and biochemical data establish that EsaR regulates its own expression by signal‐independent repression and signal‐dependent derepression. Additionally, we provide evidence that EsaR does not govern the expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis.
An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+
Authors: Guffanti Arthur A. , Wei Yi , Rood Sacha V. , Krulwich Terry A. 
Published: March 2003

SummaryMembers of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cad‐mium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.