bluuebunny/crossref_metadata_2025 huggingface dataset:
A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility
Authors: Yang Zhaomin , Geng Yongzhi , Xu Di , Kaplan Heidi B. , Shi Wenyuan
Published: March 2003

Myxococcus xanthus
cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new
M. xanthus
locus, designated
dif
for defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the
dif
locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl‐accepting chemotaxis proteins), CheW, CheY and CheA. The
dif
genes are distinct genetically and functionally from the previously identified
M. xanthus frz
chemotaxis genes, suggesting that multiple chemotaxis‐like systems are required for the developmental process of
M. xanthus
fruiting body formation. Genetic analysis and phenotypical characterization indicate that the
M. xanthus dif
locus is required for social (S) motility. This is the first report of a
M. xanthus
chemotaxis‐like signal transduction pathway that could regulate or co‐ordinate the movement of
M. xanthus cells to bring about S motility.
Masc2, a gene from Ascobolus encoding a protein with a DNA‐methyltransferase activity in vitro, is dispensable for in vivo methylation
Authors: Malagnac Fabienne , Grégoire Annie , Goyon Christophe , Rossignol Jean‐Luc , Faugeron Godeleine
Published: March 2003

We have shown previously that masc1, a gene encoding a putative C5‐DNA‐methyltransferase (MTase), was necessary for the de novo‘Methylation Induced Premeiotically’ (MIP) process and sexual reproduction in Ascobolus, whereas it was dispensable for maintenance methylation. A second MTase gene from Ascobolus, masc2, encodes a protein, Masc2, which possesses the large amino‐terminal part characteristic of eukaryotic maintenance MTases. In vitro assays have shown that Masc2 displays a methylation activity, suggesting that it might be the MTase responsible for maintenance methylation. To check its function in vivo, we engineered a disruption of the masc2 gene. The resulting mutant strains did not exhibit any particular phenotype during either vegetative growth or sexual reproduction. Neither the masc2 mutation nor the double masc1 masc2 mutation had any detectable effect upon the maintenance of the pre‐existing methylation of single gene copies previously subjected to MIP, natural retroelement‐like repeats and tandemly repeated rDNA. The masc2 mutation did not alter either MIP or the other de novo methylation process that operates in vegetative cells. Nor did it impair the meiotic process of methylation transfer. These results suggest that at least a third MTase gene responsible for maintenance and vegetative de novo methylation is present in Ascobolus.
The yeast inositol monophosphatase is a lithium‐ and sodium‐sensitive enzyme encoded by a non‐essential gene pair
Authors: Lopez Félicie , Leube Martin , Gil‐Mascarell Rosario , Navarro‐Aviñó Juan P. , Serrano Ramón
Published: March 2003

Inositol monophosphatases (IMPases) are lithium‐sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol‐1‐phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation‐extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation‐extrusion ATPase.
Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu
Authors: Lee Vincent T. , Schneewind Olaf
Published: March 2003

Gram‐negative bacteria use type III machines to inject toxic proteins into the cytosol of eukaryotic cells. Pathogenic Yersinia species export 14 Yop proteins by the type III pathway and some of these, named effector Yops, are targeted into macrophages, thereby preventing phagocytosis and allowing bacterial replication within lymphoid tissues. Hitherto, YopB/YopD were thought to insert into the plasma membrane of macrophages and to promote the import of effector Yops into the eukaryotic cytosol. We show here that the type III machines of yersiniae secrete three proteins into the extracellular milieu (YopB, YopD and YopR). Although intrabacterial YopD is required for the injection of toxins into eukaryotic cells, secreted YopB, YopD and YopR are dispensable for this process. Nevertheless, YopB, YopD and YopR are essential for the establishment of Yersinia infections in a mouse model system, suggesting that type III secretion machines function to deliver virulence factors into the extracellular milieu also.
Proteoglycan receptor binding by Neisseria gonorrhoeae MS11 is determined by the HV‐1 region of OpaA
Authors: Grant Christopher C. R. , Bos Martine P. , Belland Robert J.
Published: March 2003

The interaction of the OpaA protein of Neisseria gonorrhoeae MS11mk with heparan sulphate‐containing proteoglycan receptors on Chang conjunctiva epithelial cells was examined using isolated receptor binding and cell adherence/internalization assays. OpaA deletion proteins, in which the four surface‐exposed regions of the protein were deleted individually, and chimeric OpaA/B proteins, in which the surface‐exposed regions of the OpaA and OpaB proteins were exchanged, were expressed in N. gonorrhoeae. The recombinant deletion proteins and the chimeric OpaA/B proteins were surface exposed in the outer membrane of N. gonorrhoeae. Isolated receptor‐binding assays and Chang cell infection assays with OpaA deletion variants indicated that hypervariable region 1 was essential for the interaction of N. gonorrhoeae with the proteoglycan receptor. Expression of chimeric OpaA/B proteins confirmed the central role of hypervariable region 1 in receptor binding and demonstrated that this domain alone confers the invasive biological phenotype in a non‐heparan sulphate proteoglycan‐binding Opa protein. The other variable regions of OpaA enhanced receptor binding in the presence of region 1, but did not constitute binding domains on their own. The results indicate that proteoglycan receptor binding results from a hierarchical interaction between the variable domains of the OpaA protein of MS11mk.
Lrp binds to two regions in the dadAX promoter region of Escherichia coli to repress and activate transcription directly
Authors: Zhi Jizu , Mathew Elizabeth , Freundlich Martin
Published: March 2003

The dadAX operon is expressed by multiple promoters that are repressed by leucine‐responsive regulatory protein (Lrp) and activated by cyclic AMP‐CRP. In previous work, we found that alanine or leucine acted as inducers to antagonize Lrp repression of the three major promoters directly. Here, we identify 11 Lrp binding sites located within 350 bp of dad DNA. A mutational analysis, coupled with in vivo and in vitro transcription experiments, indicated that Lrp sites that overlap the dad promoters were involved in repression. In contrast, sites upstream of the promoters did not appear to be necessary for repression, but were required for activation by Lrp plus alanine or leucine of one of the major dad promoters, P2. This activation by alanine or leucine was not simply relief of repression, as P2 transcription from a constitutive template was increased fivefold compared with the basal level of transcription found in the absence of Lrp and the co‐activator cyclic AMP‐CRP. Alanine or leucine decreased the affinity of Lrp to repressor sites, while having little or no effect on the binding of Lrp to activator sites. This differential effect of alanine and leucine on Lrp binding helps to explain how these modifiers influence both repression and activation of the dad operon.
Serum opacity factor is a major fibronectin‐binding protein and a virulence determinant of M type 2 Streptococcus pyogenes
Authors: Courtney Harry S. , Hasty David L. , Li Yi , Chiang Herbert C. , Thacker Jenny L. , Dale James B.
Published: March 2003

Serum opacity factor (SOF) is a fibronectin‐binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by ≈35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin‐binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin‐binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF‐negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF‐negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin‐binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin‐binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.
Enzymatic and physiological properties of the tungsten‐substituted molybdenum TMAO reductase from Escherichia coli
Authors: Buc Jean , Santini Claire‐Lise , Giordani Roger , Czjzek Mirjam , Wu Long‐Fei , Giordano Gérard
Published: March 2003

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of + 130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto‐TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto‐substituted TMAO reductase (W‐TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo‐TMAO reductase (Mo‐TorA). W‐TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo‐TorA. Most importantly, the W‐TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.
FtsZ ring clusters in min and partition mutants: role of both the Min system and the nucleoid in regulating FtsZ ring localization
Authors: Yu Xuan‐Chuan , Margolin William
Published: March 2003

To understand further the role of the nucleoid and the min system in selection of the cell division site, we examined FtsZ localization in Escherichia coli cells lacking MinCDE and in parC mutants defective in chromosome segregation. More than one FtsZ ring was sometimes found in the gaps between nucleoids in min mutant filaments. These multiple FtsZ rings were more apparent in longer cells; double or triple rings were often found in the nucleoid‐free gaps in ftsI min and ftsA min double mutant filaments. Introducing a parC mutation into the ftsA min double mutant allowed the nucleoid‐free gaps to become significantly longer. These gaps often contained dramatic clusters of FtsZ rings. In contrast, filaments of the ftsA parC double mutant, which contained active MinCDE, assembled only one or two rings in most of the large nucleoid‐free gaps. These results suggest that all positions along the cell length are competent for FtsZ ring assembly, not just sites at mid‐cell or at the poles. Consistent with previous results, unsegregated nucleoids also correlated with a lack of FtsZ localization. A model is proposed in which both the inhibitory effect of the nucleoid and the regulation by MinCDE ensure that cells divide precisely at the midpoint.
Transcription‐ and translation‐dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation
Authors: Binenbaum Zoya , Parola Abraham H. , Zaritsky Arieh , Fishov Itzhak
Published: March 2003

Cell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion (‘transertion’) of nascent membrane and exported proteins. Disruption of domain structure should lead to changes in membrane dynamics. Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain. The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later. The drug‐induced decrease in membrane viscosity is energy dependent: it did not happen in KCN‐treated cells. All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion. Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand. Changes in membrane dynamics were also displayed during a nutritional shift‐down transition that causes imbalance in macromolecular syntheses. The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is ‘frozen’.
Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein‐encoding allele
Authors: Allaoui Abdelmounaaïm , François Céline , Zemzoumi Khalid , Guilvard Eliane , Ouaissi Ali
Published: March 2003

We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol–disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi‐associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene‐targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild‐type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/−) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild‐type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild‐type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP‐coupled pump, had a similar effect on wild‐type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/− single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.
Role of Lon and ClpX in the post‐translational regulation of a sigma subunit of RNA polymerase required for cellular differentiation in Bacillus subtilis
Authors: Liu Jiajian , Cosby W. Mark , Zuber Peter
Published: March 2003

The RNA polymerase sigma subunit, σH (Spo0H) of Bacillus subtilis, is essential for the transcription of genes that function in sporulation and genetic competence. Although spo0H is transcriptionally regulated by the key regulatory device that controls sporulation initiation, the Spo0 phosphorelay, there is considerable evidence implicating a mechanism of post‐translational control that governs the activity and concentration of σH. Post‐translational control of spo0H is responsible for the reduced expression of genes requiring σH under conditions of low environmental pH. It is also responsible for heightened σH activity upon relief of acid stress and during nutritional depletion. In this study, the ATP‐dependent proteases LonA and B and the regulatory ATPase ClpX were found to function in the post‐translational control of σH. Mutations in lonA and lonB result in elevated σH protein concentrations in low‐pH cultures. However, this is not sufficient to increase σH‐dependent transcription. Activation of σH‐dependent transcription upon raising medium pH and in cells undergoing sporulation requires clpX, as shown by measuring the expression of lacZ fusions that require σH for transcription and by complementation of a clpX null mutation. A hypothesis is presented that low environmental pH results in the Lon‐dependent degradation of σH, but the activity of σH in sporulating cells and in cultures at neutral pH is stimulated by a ClpX‐dependent mechanism in response to nutritional stress.
Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter
Authors: Pattery Theresa , Hernalsteens Jean‐Pierre , De Greve Henri
Published: March 2003

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron‐ and pH‐inducible lipoprotein gene sfbA, which is a component of a novel ABC‐type transporter system required for virulence. This gene is located on a 4 kb Salmonella‐specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron‐binding lipoprotein SfbA, a nucleotide‐binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild‐type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.
Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens
Authors: Jungblut P. R. , Schaible U. E. , Mollenkopf H.‐J. , Zimny‐Arndt U. , Raupach B. , Mattow J. , Halada P. , Lamer S. , Hagens K. , Kaufmann S. H. E.
Published: March 2003

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non‐virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two‐dimensional electrophoresis (2‐DE) combining non‐equilibrium pH gradient electrophoresis (NEPHGE) with SDS–PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2‐DE database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE). Silver‐stained 2‐DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.
The N‐terminus promotes oligomerization of the Escherichia coli initiator protein DnaA
Authors: Weigel Christoph , Schmidt Andrea , Seitz Harald , Tüngler Doreen , Welzeck Michaela , Messer Walter
Published: March 2003

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo‐oligomerization of DnaA via its N‐terminus (amino acid residues 1–86) is also essential for initiation. Results from solid‐phase protein‐binding assays indicate that residues 1–86 (or 1–77) of DnaA are necessary and sufficient for self interaction. Using a ‘one‐hybrid‐system’ we found that the DnaA N‐terminus can functionally replace the dimerization domain of coliphage lambda cI repressor: a λcI‐DnaA chimeric protein inhibits λ plasmid replication as efficiently as λcI repressor. DnaA derivatives with deletions in the N‐terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N‐terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N‐termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross‐talk with the nucleotide‐binding domain III or the DNA‐binding domain IV. We propose that E. coli DnaA is composed of largely independent domains — or modules — each contributing a partial, though essential, function to the proper functioning of the ‘holoprotein’.
The A‐factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus : identification of a target gene of the A‐factor receptor
Authors: Ohnishi Yasuo , Kameyama Shogo , Onaka Hiroyasu , Horinouchi Sueharu
Published: March 2003

In Streptomyces griseus, A‐factor (2‐isocapryloyl‐3R‐hydroxymethyl‐γ‐butyrolactone) at an extremely low concentration triggers streptomycin biosynthesis and cell differentiation by binding a repressor‐type receptor protein (ArpA) and dissociating it from DNA. An A‐factor‐responsive transcriptional activator (AdpA) able to bind the promoter of strR, a pathway‐specific regulatory gene responsible for transcription of other streptomycin biosynthetic genes, was purified to homogeneity and adpA was cloned by PCR on the basis of amino acid sequences of purified AdpA. adpA encoding a 405‐amino‐acid protein containing a helix‐turn‐helix DNA‐binding motif at the central region showed sequence similarity to transcriptional regulators in the AraC/XylS family. The −35 and −10 regions of the adpA promoter were found to be a target of ArpA; ArpA bound the promoter region in the absence of A‐factor and exogenous addition of A‐factor to the DNA–ArpA complex immediately released ArpA from the DNA. Consistent with this, S1 nuclease mapping showed that adpA was transcribed only in the presence of A‐factor and strR was transcribed only in the presence of intact adpA. Furthermore, adpA disruptants produced no streptomycin and overexpression of adpA caused the wild‐type S. griseus strain to produce streptomycin at an earlier growth stage in a larger amount. On the basis of these findings, we propose here a model to demonstrate how A‐factor triggers streptomycin biosynthesis at a late exponential growth stage.
Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage λ N protein and RNA
Authors: Mah Thien‐Fah , Li Joyce , Davidson Alan R. , Greenblatt Jack
Published: March 2003

The association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the efficiency of termination at intrinsic terminators. NusA is also part of the phage λ N protein‐modified antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase‐binding regions in NusA, one in the amino‐terminal 137 amino acids and the other in the carboxy‐terminal 264 amino acids; only the amino‐terminal RNA polymerase‐binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy‐terminal region of NusA is also required for interaction with N and is important for the formation of an N–NusA–nut site or N–NusA–RNA polymerase–nut site complex; the instability of complexes lacking this carboxy‐terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.
An ordered reaction mechanism for bacterial toxin acylation by the specialized acyltransferase HlyC: formation of a ternary complex with acylACP and protoxin substrates
Authors: Stanley Peter , Hyland Caroline , Koronakis Vassilis , Hughes Colin
Published: March 2003

The 110 kDa haemolysin protoxin (proHlyA) is activated in the Escherichia coli cytosol by acyl carrier protein‐dependent fatty acylation of two internal lysine residues, directed by the co‐synthesized protein HlyC. Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis–Menten analysis. The Vmax of HlyC at saturating levels of both substrates was ≈ 115 nmol acyl group min−1 mg−1 with KmacylACP of 260 nM and KmproHlyA of 27 nM, kinetic parameters sufficient to explain why in vivo HlyC is required at a concentration equimolar to proHlyA. HlyC bound the fatty acyl group from acylACP to generate an acylated HlyC intermediate that was depleted in the presence of proHlyA, but enriched in the presence of proHlyA derivatives lacking acylation target sites. HlyC was also able to bind in vivo 4′‐phosphopantetheine. Substitution of conserved amino acids that could act as putative covalent attachment sites did not prevent binding of the fatty acyl or 4′‐phosphopantetheine groups. These data and substrate variation analyses suggest that the unique acylation reaction does not involve covalent attachment of fatty acid to the acyltransferase, but rather that it proceeds via a sequential ordered Bi–Bi reaction mechanism, requiring the formation of a non‐covalent ternary acylACP–HlyC–proHlyA complex.
Residual polyadenylation in poly(A) polymerase I (pcnB ) mutants of Escherichia coli does not result from the activity encoded by the f310 gene
Authors: Mohanty Bijoy K. , Kushner Sidney R.
Published: March 2003

As extracts of poly(A) polymerase I (PAP I) deficient strains of Escherichia coli appeared to contain considerable residual polyadenylating activity, efforts were undertaken to identify a second poly(A) polymerase. Recently, a gene (f310 ) encoding the putative second poly(A) polymerase was cloned and sequenced. Here we have tested the ability of the F310 protein to add poly(A) tails in vivo by measuring total poly(A) levels in both f310 mutants and strains that overproduce F310. In addition, we have visualized poly(A) tails and examined ColE1 plasmid copy number in various genetic backgrounds. We also carried out direct biochemical measurements of AMP incorporation, using cell extracts after amplification of F310. All the data obtained indicate that F310 is not a poly(A) polymerase. Although the presence of two potential ATP binding domains in the F310 protein may account for its apparent ATP binding activity, its true biochemical function remains to be identified. In addition, we show that the f310 gene is transcribed, almost exclusively, during stationary phase from a σs promoter.
A molecular switch in SecA protein couples ATP hydrolysis to protein translocation
Authors: Karamanou Spyridoula , Vrontou Eleftheria , Sianidis George , Baud Catherine , Roos Tilmann , Kuhn Andreas , Politou Anastasia S. , Economou Anastassios
Published: March 2003

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP‐driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N‐domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C‐domain. The C‐domain binds to the N‐domain in each protomer and to the C‐domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C‐domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N‐/C‐domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY‐bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide‐regulated relative motions between the N‐domain and the C‐domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.
A folding variant of α‐lactalbumin with bactericidal activity against Streptococcus pneumoniae
Authors: Håkansson Anders , Svensson Malin , Mossberg Ann‐Kristin , Sabharwal Hemant , Linse Sara , Lazou Irene , Lönnerdal Bo , Svanborg Catharina
Published: March 2003

This study describes an α‐lactalbumin folding variant from human milk with bactericidal activity against antibiotic‐resistant and ‐susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion‐exchange matrix and eluted only with high salt. The eluted fraction showed N‐terminal and mass spectrometric identity with human milk α‐lactalbumin, but native α‐lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to α‐lactalbumin from human milk whey, but fluctuating tertiary structure. Native α‐lactalbumin could be converted to the active bactericidal form by ion‐exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram‐negative and other Gram‐positive bacteria were resistant. The folding variant of α‐lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.
Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85
Authors: Puech Virginie , Bayan Nicolas , Salim Karima , Leblon Gérard , Daffé Mamadou
Published: March 2003

Mycolic acids, long‐chain (C70–C90) α‐alkyl, β‐hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter‐chain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N‐terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild‐type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1‐inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6‐corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild‐type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1‐inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum.
IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli
Authors: Grimwade Julia E. , Ryan Valorie T. , Leonard Alan C.
Published: March 2003

In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9‐mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell‐free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non‐R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle.
The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella typhimurium virulence genes
Authors: Darwin K. Heran , Miller Virginia L.
Published: March 2003

SigD and SigE (Salmonellainvasion gene) are proteins needed for optimal invasion of Salmonella typhimurium into eukaryotic cells in vitro. SigD is a secreted protein and SigE is a putative chaperone required for SigD stability and/or secretion. SigD is secreted by a type III secretion apparatus encoded within a pathogenicity island on the Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI1). The expression of sigDE, which is not linked to SPI1, is co‐ordinately regulated with the SPI1 genes and is dependent on the transcriptional regulators SirA, HilA and InvF. These three proteins alone are unable to activate transcription from the sigD promoter in Escherichia coli, therefore it is likely that other factors are needed for expression. A screen for genes required for the expression of a sigD–lacZYA reporter fusion found a mutant with a transposon insertion in spaS, an SPI1 gene which encodes a putative inner‐membrane component of the type III secretion system. The expression of a SPI1 operon encoding a putative chaperone (SicA) and several secreted proteins (Sips B, C, D and A) was also reduced in this mutant. The regulation defect of the spaS mutant was complemented by sicA and not by spaS. Because sicA is encoded immediately downstream of spaS, the mutation in spaS was likely to be polar on the expression of sicA. In addition, a sicA disruption mutant was as defective as an invF deletion mutant for the expression of sigD, sicA and sipC reporter fusions. The introduction of plasmids encoding invF and sicA into a non‐pathogenic E. coli K‐12 strain stimulated the transcription of both a sicA– and a sigD–lacZYA promoter fusion. This result suggests that InvF and SicA are sufficient for the expression of these genes. This is the first demonstration of a positive regulatory role for a putative type III secretion system chaperone in the expression of virulence genes.
The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility
Authors: Egner Ralf , Bauer Bettina E. , Kuchler Karl
Published: March 2003

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p‐mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non‐responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p–S1360A is hypersensitive to FK506 inhibition when compared with either wild‐type Pdr5p or the non‐responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.
Antisense RNA‐mediated transcriptional attenuation: an in vitro study of plasmid pT181
Authors: Brantl Sabine , Wagner E. Gerhart H.
Published: March 2003

Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMβ1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the antisense system of plasmid pT181. The secondary structures of antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single‐round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 antisense RNA was sufficient for stable complex formation, both stem–loop structures of pT181‐RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30–50% in the absence of antisense RNA), the sense RNA of pT181 required antisense RNA for induced termination. Rate constants of formation of pT181 sense–antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10‐fold faster than stable binding.
Antagonistic control of the Escherichia coli bgl promoter by FIS and CAP in vitro
Authors: Caramel Angela , Schnetz Karin
Published: March 2003

The wild‐type Escherichia coli bgl promoter is silent in vivo but active in vitro. Silencing in vivo is directed by silencer sequences that flank the promoter, and requires nucleoid‐associated protein H‐NS and other unidentified cellular factors. Here we show that the DNA bending protein FIS is a repressor of the bgl promoter. Two FIS binding sites, centred at positions −52 and −27, overlap the CAP binding site and the −35 box respectively. FIS efficiently competes with CAP for binding to the wild‐type promoter. However, FIS does not prevent binding of RNA polymerase. It interferes with the formation of a heparin‐resistant complex and represses transcription initiation up to 40‐fold. The presence of CAP has very little effect on the FIS‐mediated repression of the wild‐type bgl promoter in vitro. However, when a bgl promoter allele was tested that carries an improved CAP binding site (which leads to activation in vivo) CAP effectively counteracted repression by FIS in vitro. These results suggest that FIS contributes to silencing of the wild‐type bgl promoter in vivo, presumably in the early exponential phase when FIS is predominantly expressed.
The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction
Authors: Shiomi Daisuke , Okumura Hisashi , Homma Michio , Kawagishi Ikuro
Published: March 2003

In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively. CheR binds to major chemoreceptors through their C‐terminal motif NWETF, which is distinct from the methylation sites. In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar. Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553. Substitution of residues N549, E551 and T552 had only a slight or little effect. The defects of the W550A and F553A mutations were suppressed by high‐ and low‐level overproduction of CheR respectively. Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain. In an in vitro assay, CheR bound to the wild‐type version but not to the mutant versions. These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR–NWETF complex. On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co‐crystal.
A folding variant of α‐lactalbumin with bactericidal activity against Streptococcus pneumoniae
Authors: Håkansson Anders , Svensson Malin , Mossberg Ann‐Kristin , Sabharwal Hemant , Linse Sara , Lazou Irene , Lönnerdal Bo , Svanborg Catharina
Published: March 2003

As the result of a typesetting error, Fig.2 in the article by Håkansson et al. on pages 589–600 of Molecular Microbiology, Volume 35, Issue 3, was unfortunately reproduced as a low‐resolution image. The correct version is reproduced here.
Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli
Authors: Hendricks E. Cale , Szerlong Heather , Hill Thomas , Kuempel Peter
Published: March 2003

We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA‐negative cells. If SOS induction was blocked by lexA3 (Ind−), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.