bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti‐anti‐σF factor SpoIIAA lead to a Spo− phenotype
Authors: Lee Chung‐Sheng , Clarkson Joanna , Shu Jwu‐Ching , Campbell Iain D. , Yudkin Michael D. 
Published: March 2003

Sigma‐F, the first sporulation‐specific transcription factor of Bacillus subtilis, is regulated by an anti‐sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti‐anti‐sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site‐directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo−. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non‐covalent complex that wild‐type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild‐type SpoIIAA‐P, but the rate of hydrolysis of SpoIIAAL61A‐P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.
The CRP–cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli
Authors: Tian Zhe‐Xian , Li Quan‐Sheng , Buck Martin , Kolb Annie , Wang Yi‐Ping 
Published: March 2003

In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a σ70‐dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen‐deficient growth conditions, glnAp1 is repressed by NtrC‐phosphate. The downstream glnAp2 promoter is σ54‐dependent and is activated by NtrC‐phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP–cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP–cAMP is observed. Site‐directed mutagenesis and deletion analysis indicate that the characterized and putative CRP‐binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP–cAMP inhibits σ54‐dependent glnAp2 strongly, by 21‐fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP–cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of γ‐glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP–cAMP activation of glnAp1 is to partially compensate for CRP–cAMP downregulation of glnAp2, allowing a low but non‐negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.
Structure of the Lrp‐regulated serA promoter of Escherichia coli K‐12
Authors: Yang Li , Lin Rong Tuan , Newman Elaine B. 
Published: March 2003

SummaryExpression of the Escherichia coli serA gene is activated in vivo by the product of the lrp gene, leucine‐responsive regulatory protein (Lrp), an effect partially reversed by L‐leucine. We show here that serA is transcribed from two promoters, P1 45 bp upstream of the translation start site, and P2 92 bp further upstream. Lrp binds to a long AT‐rich sequence from –158 to –82 from the start of the coding region, i.e. upstream of P1 and overlapping P2. It activates transcription from P1 and represses expression from P2. A second regulator, cAMP/CRP, activates P2, an effect that is largely inhibited by Lrp, such that catabolite repressor protein (Crp) and Lrp are rival activators of serA transcription.
Proteolysis and its regulation at the surface of Streptococcus pyogenes
Authors: Rasmussen Magnus , Björck Lars 
Published: March 2003

SummaryPathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus
pyogenes during infection with this important human pathogen.◊Streptococcus
pyogenes produces proteinases, and host protein‐ases are produced and released
as a result of the infection. Streptococcus pyogenes also recruits host proteinase
inhibitors to its surface, suggesting that proteolysis is tightly regulated at the
bacterial surface. We propose that the initial phase of a S. pyogenes infection
is characterized by inhibition of proteolysis and complement activity at the bacterial
surface. This is achieved mainly through binding of host proteinase inhibitors and
complement regulatory proteins to bacterial surface proteins. In a later phase of
the infection, massive proteolytic activity will release bacterial surface proteins
and degrade human tissues, thus facilitating bacterial spread. These proteolytic
events are regulated both temporally and spatially, and should influence virulence
and the outcome of S. pyogenes infections.
Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin‐producing Escherichia coli O157:H7
Authors: Barua Soumitra , Yamashino Takafumi , Hasegawa Tadao , Yokoyama Keiko , Torii Keizo , Ohta Michio 
Published: March 2003

SummaryIn general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid‐rich conditions. We found that the Shiga Toxin‐producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K‐12 strain in Luria–Bertani (LB)‐2‐morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini‐Tn5 mutagenesis and, from 50 000 colonies, five mutants that showed a clear acetic acid‐sensitive phenotype were isolated. The insertion of mini‐Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O‐polysaccharide, loss of both O‐polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O‐polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O‐polysaccharide and ECA, and normal growth was restored in organic acid‐rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O‐polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O‐polysaccharide and ECA as well as an acid resistance mechanism.
Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae
Authors: Namy Olivier , Duchateau‐Nguyen Guillemette , Rousset Jean‐Pierre 
Published: March 2003

SummaryThe efficiency of translation termination in yeast can vary several 100‐fold, depending on the context around the stop codon. We performed a computer analysis designed to identify yeast open reading frames (ORFs) containing a readthrough motif surrounding the termination codon. Eight ORFs were found to display inefficient stop codon recognition, one of which, PDE2, encodes the high‐affinity cAMP phosphodiesterase. We demonstrate that Pde2p stability is very impaired by the readthrough‐dependent extension of the protein. A 20‐fold increase in readthrough of PDE2 was observed in a [PSI+] as compared with a [psi–] strain. Consistent with this observation, an important increase in cAMP concentration was observed in suppressor backgrounds. These results provide a molecular explanation for at least some of the secondary phenotypes associated with suppressor backgrounds.
Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells
Authors: Püls Jürgen , Fischer Wolfgang , Haas Rainer 
Published: March 2003

Summary Helicobacter pylori type I strains harbour the cag pathogenicity
island (cag‐PAI), a 37 kb sequence, which encodes the components of a type
IV secretion system. CagA, the first identified effector protein of the cag‐PAI,
is translocated into eukaryotic cells and tyrosine phosphorylated (CagAP‐tyr)
by a host cell tyrosine kinase. Translocation of CagA induces the dephosphorylation
of a set of phosphorylated host cell proteins of unknown identity. CagA proteins
of independent H. pylori strains vary in sequence and thus in the number and
composition of putative tyrosine phosphorylation motifs (TPMs). The CagA protein
of H. pylori strain J99 (CagAJ99) does not carry any of three putative
tyrosine phosphorylation motifs (TPM‐A, TPM‐B or TPM‐C) predicted by the MOTIF
algorithm in CagA proteins. CagAJ99 is not tyrosine phosphorylated and
is inactive in the dephosphorylation of host cell proteins. By site‐specific mutagenesis,
we introduced a TPM‐C into CagAJ99 by replacing a single lysine with a
tyrosine. This slight modification resulted in tyrosine phosphorylation of CagAJ99
and host cell protein dephosphorylation. In contrast, the removal of the indigenous
TPM‐C from CagAP12 did not abolish its tyrosine phosphorylation, suggesting
that further phosphorylated sites are present in CagAP12. By generation
of hybrid CagA proteins, a phosphorylation of the most N‐terminal TPM‐A could be
excluded. Our data suggest that tyrosine phosphorylation at TPM‐C is sufficient,
but not exclusive, to activate translocated CagA. Activated CagAP‐tyr might either convert into a phosphatase itself or activate a cellular phosphatase to dephosphorylate cellular phosphoproteins and modulate cellular signalling cascades of the host.
Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence
Authors: Bierne Hélène , Mazmanian Sarkis K. , Trost Matthias , Pucciarelli M. Graciela , Liu Gwen , Dehoux Pierre ,   the European Listeria Genome Consortium, Jänsch Lothar , Portillo Francisco Garcia‐del , Schneewind Olaf , Cossart Pascale 
Published: March 2003

SummaryDuring infection of their hosts, Gram‐positive bac‐teria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C‐terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA
and srtB, were identified in the genome of the intracellular pathogenic bacterium
Listeria monocytogenes. Inactivation of srtA abolishes anchoring of
the invasion protein InlA to the bacterial surface. It also prevents the proper sorting
of several other peptidoglycan‐associated LPXTG proteins. Three were identified by
a mass spectrometry approach. The ΔsrtA mutant strain is defective
in entering epithelial cells, similar to a ΔinlA mutant. In contrast
to a ΔinlA mutant, the ΔsrtA mutant is impaired
for colonization of the liver and spleen after oral inoculation in mice. Thus, L.
monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG‐containing proteins that are involved in listerial infections.
Rpa12p, a conserved RNA polymerase I subunit with two functional domains
Authors: Mullem Vincent Van , Landrieux Emilie , Vandenhaute Jean , Thuriaux Pierre 
Published: March 2003

SummaryRpa12p is a subunit of RNA polymerase I formed of two zinc‐binding domains. The N‐terminal zinc region (positions 1–60) is poorly conserved from yeast to man. The C‐terminal domain contains an invariant Q.RSADE..T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N‐terminal domain fail to grow at 34°C, are sensitive to
nucleotide‐depleting drugs and become lethal in rpa14‐Δ mutants
lacking the non‐essential RNA polymerase I subunit Rpa14p. They also strongly alter
the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they
prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific
two‐hybrid interaction with the second largest subunit. In all these respects, N‐terminal
deletions behave like full deletions. In contrast, C‐terminal deletions retaining
only the first N‐terminal 60 amino acids are indistinguishable from wild type. Thus,
the N‐terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I
and is the only critical part of that subunit in vivo.
Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope
Authors: Scheuring Simon , Stahlberg Henning , Chami Mohamed , Houssin Christine , Rigaud Jean‐Louis , Engel Andreas 
Published: March 2003

SummaryBacterial surface layers (S‐layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments. Atomic force microscopy (AFM) was used to asses the S‐layer of Coryne‐bacterium

glutamicum formed of PS2 proteins that assemble into hexameric complexes within

a hexagonal lattice. Native and trypsin‐treated S‐layers were studied. Using the

AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers

were separated. All surfaces of native and protease‐digested S‐layers were imaged

at better than 1 nm lateral resolution. Difference maps of the topographies of native

and proteolysed samples revealed the location of the cleaved C‐terminal fragment

and the sidedness of the S‐layer. Because the corrugation depths determined from

images of both sides span the total thickness of the S‐layer, a three‐dimensional

reconstruction of the S‐layer could be calculated. Lattice defects visualized at

1 nm resolution revealed the molecular boundaries of PS2 proteins. The combination

of AFM imaging and single molecule force spectroscopy allowed the mechanical properties

of the Corynebacterium glutamicum S‐layer to be examined. The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.
The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP β‐lactamase
Authors: Filée Patrice , Benlafya Kamal , Delmarcelle Michaël , Moutzourelis Georgios , Frère Jean‐Marie , Brans Alain , Joris Bernard 
Published: March 2003

SummaryThe induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis
749/I BlaP β‐lactamases by β‐lactam antibiotics occurs according
to similar processes. In both bacteria, the products of the blaI and blaR1
genes share a high degree of sequence homology and act as repressors and penicillin‐sensory
transducers respectively. It has been shown in S. aureus that the BlaI repressor,
which controls the expression of BlaZ negatively, is degraded after the addition
of the inducer. In the present study, we followed the fate of BlaI during β‐lactamase
induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis
168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis
blaP, blaI and blaR1 genes. In contrast to the situation in B.
licheniformis 749/I, β‐lactamase induction in B. subtilis 168/pDML995
was not correlated with the proteolysis of BlaI. To exclude molecular variations
undetectable by SDS–PAGE, two‐dimensional gel electrophoresis was performed
with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells.
No variation in the BlaI isoelectric point was observed in induced cells, whereas
the DNA‐binding property was lost. Cross‐linking experiments with dithiobis(succimidylpropionate)
confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non‐covalent modification by a co‐activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co‐activator, our results show that the presence of penicillin stress is also required for full induction of β‐lactamase biosynthesis.
Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans
Authors: Zhang Jun , Han Gongshe , Xiang Xin 
Published: March 2003

SummaryThe multisubunit microtubule motor, cytoplasmic dynein, targets to various subcellular locations in eukaryotic cells for various functions. The cytoplasmic dynein heavy chain (HC) contains the microtubule binding and ATP binding sites for motor function, whereas the intermediate chain (IC) is implicated in the in vivo targeting of the HC. Concerning any targeting event, it is not known whether the IC has to form a complex with the HC for targeting or whether the IC can target to a site independently of the HC. In the filamentous fungus Aspergillus nidulans, the dynein HC is localized to the ends of microtubules near the hyphal tip. In this study, we demonstrate that our newly identified dynein IC in A. nidulans is also localized to microtubule ends and is required for HC’s localization to microtubule ends in living cells. With the combination of two reagents, an HC loss‐of function mutant and the green fluorescent protein (GFP)‐fused IC that retains its function, we show that the IC’s localization to microtubule ends also requires HC, suggesting that cytoplasmic dynein HC–IC complex formation is important for microtubule end targeting. In addition, we show that the HC localization is not apparently altered in the deletion mutant of NUDF, a LIS1‐like protein that interacts directly with the ATP‐binding domain of the HC. Our study suggests that, although HC–IC association is important for the targeting of dynein to microtubule ends, other essential components, such as NUDF, may interact with the targeted dynein complex to produce full motor activities in vivo.
A basic helix–loop–helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei
Authors: Borneman Anthony R. , Hynes Michael J. , Andrianopoulos Alex 
Published: March 2003

SummaryMembers of the APSES protein group are basic helix–loop–helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only
member of its genus to display a dimorphic growth transition. At 25°C, P.
marneffei grows with a filamentous morphology and produces asexual spores from
multicellular con‐idiophores. At 37°C, the filamentous morphology is replaced
by yeast cells that reproduce by fission. We have cloned and characterized an APSES
protein‐encoding gene from P. marneffei that has a high degree of similarity
to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei
showed that it is required for metula and phialide formation during conidiation but
is not required for dimorphic growth. This suggests that APSES proteins may control
processes that require budding (formation of the metulae and phialides, pseudohyphal
growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans)
but not those that require fission (dimorphic growth in P. marneffei). The
A. nidulansΔstuA mutant has defects in both conidiation
and mating. The P. marneffei stuA gene was capable of complementing the conidiation
defect but could only inefficiently complement the sexual defects of the A. nidulans
mutant. This suggests that the P. marneffei gene, which comes from an asexual
species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.
Nitrate and the GATA factor AreA are necessary for in vivo binding of NirA, the pathway‐specific transcriptional activator of Aspergillus nidulans
Authors: Narendja Frank , Goller Sabine P. , Wolschek Markus , Strauss Joseph 
Published: March 2003

SummaryIn Aspergillus nidulans, the genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), are transcribed divergently from a common promoter region of 1200 basepairs. We have previously characterized the relevant cis‐acting elements for the two synergistically acting transcriptional activators NirA and AreA. We have further shown that AreA is constitutively bound to a central cluster of four GATA sites, and is involved in opening the chromatin structure over the promoter region thus making additional cis‐acting binding sites accessible. Here we show that the asymmetric mode of NirA–DNA interaction determined in vitro is also found in vivo. Binding of the NirA transactivator is not constitutive as in other binuclear C6‐Zn2+‐cluster proteins but depends on nitrate induction and, additionally, on the presence of a wild‐type areA allele. Dissecting the role of AreA further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly exert its effect on NirA via inducer exclusion. We have tested this possibility in a strain accumulating nitrate in the absence of areA. We found that in such a strain the intracellular presence of inducer is not sufficient to promote either chromatin rearrangement or NirA binding, implying that both processes are directly dependent on AreA.
The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components
Authors: Robson Scott A. , Michie Katharine A. , Mackay Joel P. , Harry Elizabeth , King Glenn F. 
Published: March 2003

SummaryThe bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the ‘septasome’) that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two‐hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdepend‐ence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.