bluuebunny/crossref_metadata_2025 huggingface dataset:
Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice
Authors: Stone Barbara J. , Miller Virginia L.
Published: March 2004

The ability of Salmonella to invade tissue culture cells is correlated with virulence. Therefore, the tissue culture invasion model has been used extensively to study this process and to identify the bacterial genes involved and their products. Described here is the further characterization of a Salmonella enteritidis mutant (SM6T) originally identified as non‐invasive for tissue culture cells. A chromosomal DNA fragment complementing this defect was cloned and sequenced. The derived protein sequence is 89% identical to TolC from Escherichia coli, an outer membrane protein required for the signal peptide‐independent transport of α‐haemolysin and colicin V. Therefore, sinA was renamed tolC and is referred to in this text as tolCs to distinguish it from tolC of E. coli TolCs and TolC are functionally similar since tolC can complement the invasion‐defective phenotype of a tolCs mutant, and tolCs is required for export of α‐haemolysin by Salmonella. The tolCs mutant is avirulent for mice when administered by the oral route, suggesting that the gene is important for virulence. Further characterization of the tolCs mutant indicated that like tolC mutants it is more sensitive than the wild‐type strain to various detergents, antibiotics and dyes. This mutant is more sensitive to Triton X‐100 only when associated with the monolayer, and the invasion‐defective phenotype appears to be an artifact of this sensitivity. In addition, the tolCs mutant is more sensitive to the bactericidal activity of human serum. Therefore, the avirulent phenotype could be the result of an inability to secrete a necessary virulence factor, or an increased sensitivity to complement and detergents as a result of a subtle alteration in the lipopolysaccharide (LPS) associated with tolC mutations.
An rpoE‐like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep‐sea bacterium Photobacterium sp. strain SS9
Authors: Chi E. , Bartlett D.H.
Published: March 2004

Many deep‐sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro‐ and baro‐ adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1–3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma factors activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE, we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro‐ and psychro‐ sensitive. ORF3 mutant revertants that displayed enhanced high‐pressure growth also displayed concomitant enhanced low‐temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high‐pressure and cold‐temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro‐ and psychro‐adaptation.
Localized denaturation of oriT DNA within relaxosomes of the broad‐host‐range plasmid R1162
Authors: Zhang Shuyu , Meyer Richard J.
Published: March 2004

The broad‐host‐range, multicopy plasmid R1162 is efficiently mobilized during conjugation by the self‐transmissible plasmid R751. The relaxosome, a complex of plasmid DNA and R1162‐encoded proteins, forms at the origin of transfer (oriT) and is required for mobilization. Transfer is initiated by strand‐ and site‐specific nicking of the DNA within this structure. We show by probing with potassium permanganate that oriT DNA is locally melted within the relaxosome, in the region from the inverted repeat to the site that is nicked. Mutations in this region of oriT, and in genes encoding the protein components of the relaxosome, affect both nicking and melting of the DNA. The nicking protein in the relaxosome is MobA, which also ligates the transferred linear, single strand at the termination of a round of transfer. We propose that there is an underlying similarity in the substrates for these two MobA‐dependent, DNA‐processing reactions. We also show that MobA has an additional role in transfer, beyond the nicking and resealing of oriT DNA.
Developmental control of the heat‐shock stress regulon in Streptomyces coelicolor
Authors: Puglia Anna Maria , Vohradsky Jiri , Thompson Charles J.
Published: March 2004

In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat‐shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two‐dimensional gels and analysed using advanced statistical methods. The responses of all heat‐shock proteins (HSPs) were highly dependent upon the growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the levels of thermal induction attained were closely related to growth stage‐determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.
Antisense RNA regulation of the fatB iron transport protein gene in Vibrio anguillarum
Authors: Waldbeser Lillian S. , Chen Qian , Crosa Jorge H.
Published: March 2004

We have recently identified an antisense RNA (RNAα) that regulates the expression of the fatA iron transport gene encoding the outer membrane receptor for the iron‐anguibactin complex. In this work, we demonstrate that RNAα also inhibits the expression of fatB, which encodes a 35 kDa iron transport protein and has domains homologous to other periplasmic transport proteins. The expression of fatA and fatB is repressed under iron‐rich conditions, in which RNAα is induced. RNAα is homologous to two‐thirds of the coding region of fatB. By cloning RNAα coding sequences immediately downstream of a tet promoter, we were able to obtain constitutive expression of the antisense RNA. The cloned region contains approximately 83% of the 650 nucleotide RNAα and is complementary to only 51% of the fatB mRNA but is still capable of causing a repression of the expression of the fatB gene. Our results in this work demonstrate that RNAα probably affects the stability of the fatB‐specific mRNA.
The deletion of 70 amino acids near the N‐terminal end of the sucrose‐specific porin ScrY causes its functional similarity to LamB in vivo and in vitro
Authors: Schülein Katrin , Andersen Christian , Benz Roland
Published: March 2004

A deletion mutant ScrΔ3‐73 of the sucrose‐specific porin ScrY was constructed in which 70 amino acids of the mature protein were deleted near the N‐terminal end. ScrYΔ3‐72 was still able to oligomerize and inserted properly into the outer membrane of an Escherichia coli strain. The protein was isolated and purified by standard procedures. The mutant protein showed, in contrast to wild‐type ScrY, a tight association with the murein. Reconstitution experiments with artificial lipid bilayer membranes demonstrated that ScrYΔ3‐72 produced defined, cation‐selective channels in planar lipid bilayers. Its single‐channel conductance was reduced to about half of the value of wild‐type ScrY. The deletion had a relatively small influence on the stability constants for carbohydrate binding. However, in contrast to wild‐type ScrY, [14C]‐maltopentaose was efficiently taken up into whole E. coli cells containing ScrYΔ3‐72. The sequence of the N‐terminus of mature ScrY was identified as starting with glutamine 23. The possible structure of ScrY and ScrYΔ3‐72 in the outer membrane is discussed.
Rearrangements in the staphylococcal β‐lactamase‐encoding plasmid, plP1066, including a DNA inversion that generates two alternative transposons
Authors: Derbise A. , Dyke K.G.H. , El Solh N.
Published: March 2004

The plasmid plP1066, harboured by a methicillin‐resistant Staphylococcus aureus strain isolated in France, carries genes specifying β‐lactamase. This plasmid undergoes numerous rearrangements. One of these was an insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn5404, carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn552. These were: p480 encoding a probable transposase, p271 encoding a putative ATP‐binding protein, binL encoding a resolvase, and a resolution site, resL. In addition, Tn5404 carried aminoglycoside‐resistance genes (aphA, str) and the insertion sequence IS1181. Tn5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn552, and was flanked by 6 bp direct repeats. Insertion of Tn5404 close to resR and to the structural and regulatory β‐lactamase genes (blaZ, blal, blaR1) of plP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites (resR, resL). This invertible segment, which carried p480, p271 and binL, generated Tn552 or Tn5404, depending on its orientation. Thus, these two transposons share their transposition and resolution systems.
Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells
Authors: Hermant Daniel , Ménard Robert , Arricau Nathalie , Parsot Claude , Popoff Michel Y.
Published: March 2004

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA, was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid‐borne ipa operon. Expression of sipE and sipB in a Shigella non‐invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.
Transcriptional regulation of bioluminesence genes from Vibrio fischeri
Authors: Sitnikov Dmitry M. , Schineller Jeffrey B. , Baldwin Thomas O.
Published: March 2004

The phenomenon of cell‐density‐dependent control of gene expression, called autoinduction, has long been a subject of interest and investigation in bioluminescent marine bacteria. It is now becoming clear that many other bacteria, including animal and plant pathogens, use an autoinduction mechanism to regulate a variety of functions. Cell‐density‐dependent gene expression provides an excellent example of multicellular behaviour in the prokaryotic kingdom where a single cell is able to communicate and sense when a minimal population unit, a ‘quorum’ of bacteria, is achieved in order for certain behaviour of the population to be performed efficiently. Regulation of bacterial bioluminescence has been studied for many years and represents the best model system for understanding the mechanism of cell‐density‐dependent gene expression. This review will focus on transcriptional regulation of the Vibrio fischeri luminescence genes emphasizing the role of the transcriptional activator LuxR and possible autoinduction mechanisms that occur in E. coli Alternative views and opinions regarding the molecular details of the autoinduction mechanism will be discussed.
Distinct transcriptional regulation of a gene coding for a mitochondrial protein in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis despite similar promoter structures
Authors: Mulder Wietse , Scholten Inge H.J.M. , Grivell Leslie A.
Published: March 2004

In Saccharomyces cerevisiae transcription of QCR8, encoding subunit VIII of the mitochondrial ubiquinol cytochrome c oxidoreductase, is subject to glucose repression, whereas in the distantly related yeast Kluyveromyces lactis it is not. The homologous promoter regions lack overall DNA‐sequence identity, but do share binding sites for the transcription factors Abf1p, Cpf1p and Hap2/3/4p. For S. cerevisiae it has been established that these factors are involved in growth and carbon source control of nuclear genes encoding mitochondrial proteins. Here we present experimental evidence that K. lactis counterparts of Abf1p and Cpf1p bind independently to the KIQCR8 promoter. The presence of the KIHap2/3/4p binding site enhances binding of KIAbf1p, strongly suggesting that the KIHap2/3/4p complex stabilizes binding of KIAbf1p. In reciprocal exchanges of the QCR8 genes between S. cerevisiae and K. lactis, overall regulation of transcription was found to be species specific. In contrast to S. cerevisiae, additional elements and factors in K. lactis, distinct from Abf1p, Cpf1p and Hap2/3/4p, are able to activate transcription of the QCR8 gene during both fermentative and non‐fermentative growth, as well as to induce transcription during growth on non‐fermentable carbon sources. In both yeasts, Abf1p is involved in transcriptional activation under both fermentative and non‐fermentative growth conditions. Hap2/3/4p plays a minor activational role during fermentative growth, but is mainly responsible for transcriptional induction under non‐fermentative growth conditions. Under these latter conditions Abf1p and Hap2/3/4p activate transcription synergistically.
The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio
Authors: Skarstad Kirsten , Wold Sture
Published: March 2004

The DnaC protein is required for loading the DnaB helicase at oriC. Thus DnaC promotes the formation of the pre‐replication complex, but must leave the complex in order for the DnaB protein to function as a helicase. In vitro, a slight excess of DnaC inhibits the movement of replication forks by inhibiting DnaB helicase activity (Allen and Kornberg, 1991). Here we show that inhibition of DNA replication by excess DnaC also occurs in vivo. The rate of replication‐fork movement was measured by flow cytometry. Initiation of replication was inhibited with rifampicin and the rate of fork movement monitored during replication run‐out by measuring the increase in the fraction of the cell population with fully replicated chromosomes. The replication rate was inversely related to the amount of excess DnaC protein. Initiation of replication was also inhibited. Co‐overexpression of DnaB protein alleviated the inhibition of replication caused by moderate excess of DnaC. The results show that DnaC interacts with replication forks during elongation in vivo, probably by binding to DnaB and inhibiting its helicase activity. Therefore, the ratio of DnaC to DnaB and the affinity of DnaC for a helicase hexamer at an established replication fork are of great importance for the rate of replication fork movement also in vivo.
Characterization of domains in the yeast MAP kinase Slt2 (Mpk1) required for functional activity and in vivo interaction with protein kinases Mkk1 and Mkk2
Authors: Soler M. , Plovins A. , Martín H. , Molina M. , Nombela C.
Published: March 2004

MKK1/MKK2 and SLT2 (MPK1) are three Saccharomyces cerevisiae genes, coding for protein kinases, that have been postulated to act sequentially as part of the Pkc1p signalling pathway, a phosphorylation cascade essential for cell integrity. By using the ‘two‐hybrid system’ and co‐purification experiments on glutathione‐agarose beads, we have shown that Slt2p interacts in vivo and in vitro with both Mkk1p and Mkk2p, thus confirming a previous suggestion based on epistasis experiments of the corresponding genes. Plasmid constructs of the SLT2 gene, deleted in the whole C‐terminal non‐kinase region or part of it, and therefore containing all of the conserved kinase subdomains, were still functional in complementation of the slt2 lytic phenotype and in vivo interaction with Mkk1p and Mkk2p. In contrast, the Slt2p C‐terminal domain (162 residues) that carries a glutamine‐rich fragment followed by a 16 polyglutamine tract, was shown to be dispensable for complementation and in vivo association with Mkk1p and Mkk2p. We have also demonstrated that the N‐terminal putative regulatory domain of these two MAP kinase activators is the main region involved in the interaction with Slt2p.
Gene‐regulatory modules In Escherichia coli: nucleoprotein complexes formed by cAMP‐CRP and CytR at the nupG promoter
Authors: Pedersen Henrik , Dall Jens , Dandanell Gert , Valentin‐Hansen Poul
Published: March 2004

Repression by CytR depends on the formation of nucleoprotein complexes in which the CytR repressor and the cAMP‐CRP activator complex bind co‐operatively to the DNA. Transcription initiation from CytR‐regulated promoters requires cAMP‐CRP; therefore, the cAMP‐CRP complex functions both as an activator and as a co‐repressor in these promoters. Another interesting aspect of the CytR regulon is that each promoter appears to have individual features. Therefore, structural and functional rules governing the formation of repression and activation complexes in one promoter may not be valid for other promoters of the CytR regulon. Here we show that the Escherichia colinupG gene contains one CytR‐ and four CRP‐binding sites in the control region. Notably, the architecture of the CytR binding site is different from previously described targets. In addition, the CytR repressor triggers a DNA repositioning of a cAMP‐CRP complex in the −35 region upon binding to its operator. Thus, formation of the repression and activation complexes at the nupG promoter involves different subsets of CRP‐binding sites. These findings show that the bacterium uses positive and negative regulatory modules to differentially control the expression of CytR‐ and cAMP‐CRP‐regulated genes.
High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili
Authors: Marceau Michael , Beretti Jean‐Luc , Nassif Xavier
Published: March 2004

Pili are indispensable in adhesion of encapsulated Neisseria meningitidis (MC) to eukaryotic cells. Intrastrain variability with respect to the degree of adhesion is the result of pilin antigenic variation. We have localized the region responsible for this variability to the 20‐amino‐acid hypervariable domain of pilin. The replacement of an aspartic acid, located in the hypervariable region of a low‐adhesive variant by a lysine restored high adhesiveness. To assess whether hyperadhesiveness confered by some pilin variants was related to the generation of a new pilus‐associated ligand, high‐ and low‐adhesive variants were purified. In a first step, low‐ and high‐adhesive pilins were fused to maltose binding protein (MBP). These hybrid proteins bound epithelial cells with the same affinity. Truncated MBP pilin fusions identified a cell‐binding domain within the 77 residues of the N‐terminal end of mature pilin. This region of the protein is common to low‐ and high‐adhesive derivatives used in this work, thus eliminating the possibility that high adhesiveness confered by some pilin variants was because of the generation of a new pilus‐associated ligand. Electron‐microscopic examination showed that low‐adhesive derivatives expressed long and distinct pili and adhered as single cells. In contrast, pili of derivatives expressing high‐adhesive pilins, either wild type or mutagenized from the low‐adhesive variant, formed large bundles which bound bacteria and caused them to grow as colonies on infected mono‐layers. These data demonstrate that aggregative pili promote high adhesiveness of encapsulated MC.
Opa‐typing: a high‐resolution tool for studying the epidemiology of gonorrhoea
Authors: O'Rourke Maria , Ison Catherine A. , Renton Adrian M. , Spratt Brian G.
Published: March 2004

A single gonococcus possesses a family of 11 distinct and highly variable opa genes. The extensive variation and rapid evolution of the opa gene repertoire has been exploited to provide a high‐resolution typing method for studies of the short‐term transmission of gonorrhoea. The 11 opa genes are amplified with a single pair of primers by the polymerase chain reaction, digested with frequently‐cutting restriction enzymes, and the fragments are fractionated on polyacrylamide to provide an opa‐type. The method appeared to be highly discriminatory as the opa‐types of gonococci, isolated world‐wide over the last 30 years, were all different. Opa‐typing discriminated between isolates of the same auxotype/serovar class. Similarly, there were 41 opa‐types among 43 consecutive isolates from a sexually transmitted disease (STD) clinic. The two pairs of isolates from this clinic that gave the same opa‐types were identical by other criteria and may have been from unsuspected sexual contacts. With one minor exception, identical opa‐types were obtained from gonococci recovered from known sexual contacts. These results suggest that variation in the family of 11 opa genes evolves so rapidly that the opa‐types of gonococci are distinguishable, unless the isolates are from sexual contacts or a short chain of disease transmission. The identification of gonococci with identical opa‐types is therefore believed to be a good indicator that the individuals from which they were recovered were sexual partners, or part of a short chain of disease transmission.
Mutational analysis reveals dispensability of the N‐terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression
Authors: Langdon Tim , Sheerins Alan , Ravagnani Adriana , Gielkens Marco , Caddik Mark X. , Arst Herbert N.
Published: March 2004

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT—PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C‐terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon‐encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.
Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative‐stress regulatory gene:implications for sensitivity to isoniazid
Authors: Deretic V. , Philipp W. , Dhandayuthapani S. , Mudd M.H. , Curcic R. , Garbe T. , Heym B. , Via L.E. , Cole S.T.
Published: March 2004

The systems participating in detoxification of reactive oxygen intermediates in Mycobacterium tuberculosis are believed to play a dual role in the biology of this highly adapted human pathogen: (i) they may contribute to the survival of this bacterium in the host; and (ii) alterations in the gene encoding catalase/peroxidase have been linked to this organism's resistance to the front‐line antituberculosis drug isoniazid. These relationships prompted us to extend investigations of the oxidative‐stress‐response systems in M. tuberculosis by analysing the alkyl hydroperoxide reductase gene ahpC and its putative regulator oxyR. Surprisingly, the oxyR gene was found to be inactivated by multiple lesions in M. tuberculosis H37Rv. These alterations were observed in all M. tuberculosis strains tested, and in members of the M. tuberculosis complex: Mycobacterium bovis BCG, Mycobacterium africanum, and Mycobacterium microti. The corresponding region carrying these genes in Mycobacterium leprae, an organism not sensitive to isoniazid, has a complete oxyR gene divergently transcribed from ahpC. An increase in minimal inhibitory concentration for isoniazid was observed upon transformation of M. tuberculosis H37Rv with cosmids carrying the oxyR—ahpC region of M. leprae. In keeping with the observed inactivation of oxyR, transcriptional activity of the corresponding region in M. tuberculosis was an order of magnitude lower than that of the oxyR gene from M. leprae. While the loss of this putative regulator of oxidative‐stress response in M. tuberculosis is paradoxical considering the fact that survival in host macrophages is regarded as a critical feature of this pathogen, it offers a partial explanation for the exquisite sensitivity of M. tuberculosis to isoniazid.
Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages
Authors: Dhandayuthapani S. , Via L.E. , Thomas C.A. , Horowitz P.M. , Deretic D. , Deretic V.
Published: March 2004

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co‐factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle‐plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress‐response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two‐component signal‐transduction systems; (iii) hsp60, a previously characterized heat‐shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence‐activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA‐gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.
Green fluorescent protein as a new expression marker in mycobacteria
Authors: Kremer Laurent , Baulard Alain , Estaquier Jérôme , Poulain‐Godefroy Odile , Locht Camille
Published: March 2004

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM‐11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long‐wave u.v. light. In mycobacteria containing a pGFM‐11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter‐probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM‐11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co‐factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free‐living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.
Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar‐specific nodulatlon of soybean by Rhizobium fredii USDA257
Authors: Kovács László G. , Balatti Pedro A. , Krishnan Hari B. , Pueppke Steven G.
Published: March 2004

Rhizobium fredii is a nitrogen‐fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host‐specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert and orientation‐specific expression of two genes, noIX and nolW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.
Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation
Authors: Shankar Sandeep , Schlictman David , Chakrabarty A.M.
Published: March 2004

We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa. AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa. One of the E. coli genes, rnk, encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA, encodes the stringent starvation protein, a regulatory protein involved in stationary‐phase regulation and the stringent response of E. coli. While both rnk and sspA restored alginate production to the P. aeruginosaalgR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli. We find that a mutation in rnk drastically reduces the level of Ndk in E. coli. A mutation in sspA, however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti‐Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.
Asymmetric distribution of the Listeria monocytogenes ActA protein is required and sufficient to direct actin‐based motility
Authors: Smith Gregory A. , Portnoy Daniel A. , Theriot Julie A.
Published: March 2004

Listeria monocytogenes is a Gram‐positive facultative intracytoplasmic bacterial pathogen that exhibits rapid actin‐based motility in eukaryotic cells and in cell‐free cytoplasmic extracts. The protein product of the actA gene is required for bacterial movement and is normally expressed in a polarized fashion on the bacterial surface. Here we demonstrate that the ActA protein is sufficient to direct motility in the absence of other L. monocytogenes gene products, and that polarized localization of the protein is required for efficient unidirectional movement. We have engineered a fusion protein combining ActA with the C‐terminal domain of the LytA protein of Streptococcus pneumoniae, which mediates high‐affinity binding to DEAE‐cellulose and to choline moieties present in the S. pneumoniae cell wall. DEAE‐cellulose fragments or S. pneumoniae coated uniformly with the ActA/LytA fusion protein nucleate actin filament growth in cytoplasmic extracts, but do not move efficiently. However, when ActA/LytA‐coated S. pneumoniae is grown to polarize the distribution of the fusion protein, the bacteria exhibit unidirectional actin‐based movement similar to the normal movement of L. monocytogenes.
Specific recognition of the Bacillus subtilis gnt cis‐acting catabolite‐responsive element by a protein complex formed between CcpA and seryl‐phosphorylated HPr
Authors: Fujita Yasutaro , Miwa Yasuhiko , Galinier Anne , Deutscher Josef
Published: March 2004

Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis‐acting catabolite‐responsive element (ORE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR‐Lacl family of bacterial transcriptional repressors/activators, and the seryl‐phosphorylated form of HPr, a phosphocarrier protein of the phosphoenol‐pyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P‐ser‐HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to Tat position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P‐ser‐HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P‐ser‐HPr.
Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides a bacterium with a unidirectional, stop‐start flagellum
Authors: Shah Deepan S.H. , Sockett R. Elizabeth
Published: March 2004

Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re‐orienting randomly by Brownian motion when flagellar rotation tops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli, a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli. MotA is thought to be a proton ‘pore’ involved in converting proton‐motive force into flagellar rotation. Several potential proton‐binding amino acids were conserved between putative membrane‐spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB, Examination of the upstream DNA sequence showed only one putative promoter‐like sequence which resembled a σ54‐ type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of he R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.
Binding of the TorR regulator to cis‐acting direct repeats activates tor operon expression
Authors: Simon Gwénola , Jourlin Cécile , Ansaldi Mireille , Pascal Marie‐Claire , Chippaux Marc , Méjean Vincent
Published: March 2004

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N‐oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC′‐lacZ fusion dramatically reduces TorR‐dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel‐retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the −35 promoter sequence and includes the third tor box (box3); the last is found downstream from the −35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments.

We propose a model in which multiple binding sites (i.e. the tor boxes) contribute to the formation of a nucleoprotein complex, but only one particular proximal site positions TorR properly so that it interacts with RNA polymerase.
Participation of the purine repressor in control of the carbamoylphosphate synthetase operon in Salmonella typhimurium
Authors: Lu Chung‐Dar , Katzif Samuel , Abdelal Ahmed T.
Published: March 2004

Previous work has shown that the carAB operon of Salmonella typhimurium is transcribed from tandem promoters, P1 and P2, that are negatively controlled by pyrimidines and arginine, respectively. The results reported here show that purines also negatively control expression of carAB and that this effect is absent in a purR::Tn10 derivative. Primer‐extension experiments established that the purine effect is exerted at P1, thus redefining this promoter as sensitive to both purines and pyrimidines. The results of gel‐retardation experiments as well as DNase I and premethylation footprintings indicate that the purine repressor interacts with a PUR box 85 bp upstream of P1. Modification of this PUR box by site‐directed mutagenesis abolishes the repression by purines in a carA::lacZ fusion, confirming that this box functions in vivo in purine control of carAB expression.
Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin‐producing organism, Streptomyces pristinaespiralis
Authors: Blanc Veronique , Salah‐Bey Khadidja , Folcher Marc , Thompson Charles J.
Published: March 2004

A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analysed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription‐translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted −10 and −35 hexamers which were somewhat similar to Streptomyces vegetative‐type promoters.
Stress‐activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli
Authors: Salah‐Bey Khadidja , Blanc Veronique , Thompson Charles J.
Published: March 2004

A promoter which controls expression of the pristinamycin multidrug resistance gene (ptr) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp. and Escherichia coli. In S. pristinaspiralis, the ptr promoter (Pptr) was induced by pristinamycin I (PI) or pristinamycin II (PII). Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes. Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on. In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD. In E. coli, Pptr cloned in the bifunctional promoter probe vector plJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer. Finally, gel‐retardation studies demonstrated a Pptr‐binding protein in S. lividans (where its activity was PI‐inducible), S. coelicolor and S. pristinaespiralis. The fact that this activity was not detected in E. coli suggested the existence of another regulatory system perhaps also present in Streptomyces.
Identification by in vitro complementation of regions required for cell‐invasive activity of Bordetella pertussis adenylate cyclase toxin
Authors: Iwaki Masaaki , Ullmann Agnes , Šebo Peter
Published: March 2004

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706‐residue protein composed of an amino‐terminal adenylate cyclase (AC) domain linked to a 1300‐residue channel‐forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell‐binding domain and that structural integrity and co‐operation of all domains, as well as the post‐translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with non‐overlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.
Potassium channel activation by glutathione‐S‐conjugates in Escherichia coli: protection against methylglyoxal is mediated by cytoplasmic acidification
Authors: Ferguson Gail P. , McLaggan Debra , Booth Ian R.
Published: March 2004

Escherichia coli possesses two glutathione‐gated potassium channels, KefB and KefC, that are activated by glutathione‐S‐conjugates formed with methylglyoxal. We demonstrate that activation of the channels leads to cytoplasmic acidification and that this protects cells during electrophilic attack. Further, we demonstrate that mutants lacking the channels can be protected against the lethal effects of methylglyoxal by acidification of the cytoplasm with a weak acid. The degree of protection is determined by the absolute value of the pHi and the time at which acidification takes place. Alterations in the pHi do not accelerate the rate of detoxification of methylglyoxal. The mechanism by which methylglyoxal causes cell death and the implications for pHi‐mediated resistance to methylglyoxal are discussed.