bluuebunny/crossref_metadata_2025 huggingface dataset:
Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis
Authors: Schaller Martin , Korting Hans C. , Schäfer Wilhelm , Bastert Janine , Chen WenChieh , Hube Bernhard
Published: March 2003

Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expression of individual SAP genes has been shown previously in a model of oral candidosis based on reconstituted human epithelium (RHE), and in the oral cavity of patients. In this study, the ultrastructural localization of distinct groups of Sap isoenzymes expressed during RHE infection was shown by immunoelectron microscopy using specific polyclonal antibodies directed against the gene products of SAP1‐3 and SAP4‐6. Large amounts of Sap1‐3 antigen were found within C. albicans yeast and hyphal cell walls, often predominantly in close contact with epithelial cells, whereas lower quantities of Sap4‐6 were detected in hyphal cells. To elucidate the relevance of the expressed Saps during oral infections, we examined the effect of the aspartic proteinase inhibitor, pepstatin A, during infection of the RHE. The extent of lesions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Δsap1, Δsap2, Δsap3, Δsap4‐6 and three Δsap1/3 double mutants were examined. Although tissue damage was not blocked completely with these mutants, an attenuated phenotype was observed for each of the single sap null mutants, and was more strongly attenuated in the Δsap1/3 double null mutants. In contrast, the lesions caused by the Δsap4‐6 triple mutant were at least as severe as those caused by SC5314. During infection with the mutants, we observed that the SAP gene expression pattern of the Δsap1 and the Δsap1/3 mutants was altered in comparison with the wild‐type strain. Expression of SAP5 was observed only during infection with the Δsap1/3 mutant, whereas upregulation of SAP2 and SAP8 transcripts was observed in the Δsap1 and the Δsap1/3 mutants. These results suggest that Sap1‐3, but not Sap4‐6, contribute to tissue damage in this model. Furthermore, C. albicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.
Gene vanXYC encodes d,d ‐dipeptidase (VanX) and d,d‐carboxypeptidase (VanY) activities in vancomycin‐resistant Enterococcus gallinarum BM4174
Authors: Reynolds Peter E. , Arias Cesar A. , Courvalin Patrice
Published: March 2003

VanX and VanY have strict d,d‐dipeptidase and d,d‐carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in d‐alanyl‐d‐alanine (d‐Ala‐d‐Ala) in glycopeptide‐resistant enterococci in which the C‐terminal d‐Ala residue has been replaced by d‐lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in d‐Ala‐d‐serine (d‐Ala‐d‐Ser) essential for VanC‐type vancomycin resistance. Insertional inactivation of the vanC‐1 gene encoding the ligase that catalyses synthesis of d‐Ala‐d‐Ser has a polar effect on both d,d‐dipeptidase and d,d‐carboxypeptidase activities. The open reading frame downstream from vanC‐1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX‐ and VanY‐type enzymes. It had very low dipeptidase activity against d‐Ala‐d‐Ser, unlike VanX, and no activity against UDP‐MurNAc‐pentapeptide[d‐Ser], unlike VanY. The introduction of plasmid pAT708(vanC‐1,XYC) or pAT717(vanXYC) into vancomycin‐susceptible Enterococcus faecalis JH2‐2 conferred low‐level vancomycin resistance only when d‐Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2‐2 and JH2‐2(pAT708) and JH2‐2(pAT717) indicated that the function of VanXYC was hydrolysis of d‐Ala‐d‐Ala and removal of d‐Ala from UDP‐MurNAc‐pentapeptide[d‐Ala]. VanC‐1 and VanXYC were essential, but not sufficient, for vancomycin resistance.
Time‐lapsed confocal microscopy reveals temporal and spatial expression of the lysine ɛ‐aminotransferase gene in Streptomyces clavuligerus
Authors: Han Lei , Khetan Anurag , Hu Wei‐Shou , Sherman David H.
Published: March 2003

To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine ɛ‐aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild‐type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT–Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild‐type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time‐lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.
Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator
Authors: Karls Russell K. , Wolf Jennifer R. , Donohue Timothy J.
Published: March 2003

The Rhodobacter sphaeroides photosynthesis response regulator, PrrA, positively regulates cycA P2 expression. Deletion analysis has identified sequences within 73 bp upstream of the transcription initiation site that are required for the activation of cycA P2 by PrrA. A mutant form of the Rhodobacter capsulatus PrrA homologue, whose activity is independent of phosphorylation (RegA*), protects an ≈ 26 bp region of cycA P2 that is centred at ≈ −50 from DNase digestion, and activates transcription of a mutant −14T promoter with increased activity when using either R. sphaeroides RNA polymerase or Escherichia coli Eσ70. A 4 bp target site mutation that eliminated DNA binding and transcription activation by RegA*in vitro also abolished PrrA activation of cycA P2 transcription in vivo, indicating that this region contains a PrrA binding site. By analysing the behaviour of the −14T mutant cycA P2 promoter in vivo, we also found that PrrA uses the same target site to activate expression in both the presence and the absence of O2. However, the extent of transcription activation by PrrA at cycA P2 in vivo is greater under anaerobic conditions.
Spontaneous tandem amplification and deletion of the Shiga toxin operon in Shigella dysenteriae 1
Authors: McDonough Marie A. , Butterton Joan R.
Published: March 2003

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin‐producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage‐encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate‐containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx‐encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.
Identification of dipeptide repeats and a cell wall sorting signal in the fimbriae‐associated adhesin, Fap1, of Streptococcus parasanguis
Authors: Wu Hui , Fives‐Taylor Paula M.
Published: March 2003

Fap1, a fimbriae‐associated protein, is involved in fimbriae assembly and adhesion of Streptococcus parasanguis FW213 (Wu et al., 1998). In this study, the sequence of the fap1 gene was resolved using a primer island transposition system. Sequence analysis indicated that fap1 was composed of 7659 nucleotides. The predicted Fap1 protein contains an unusually long signal sequence (50 amino acid residues), a cell wall sorting signal and two repeat regions. Repeat regions I and II have a similar dipeptide composition (E/V/I)S, composed of 28 and 1000 repeats respectively. The two regions combined accounted for 80% of the Fap1 coding region. The experimental amino acid composition and isoelectric point (pI) of Fap1 were similar to that predicted from the deduced Fap1 protein. Results of Northern analyses revealed that the fap1 open reading frame (ORF) was transcribed as a 7.8 kb monocistronic message. Insertional inactivation at the 3′ end, downstream of the fap1 ORF, did not affect Fap1, fimbrial expression or bacterial adhesion. Insertional inactivation of fap1 immediately upstream of the repeat region II abolished expression of Fap1 and fimbriae, and was concurrent with a diminution in adhesion of FW213. Inactivation of the cell wall sorting signal of fap1 also eliminated long fimbrial formation and reduced the ability of FW213 to bind to SHA. Fap1 was no longer anchored on the cell surface. Large quantities of truncated Fap1 were found in the growth medium instead. These results suggest that the fap1 ORF alone is sufficient to support Fap1 expression and adhesion, and demonstrate that anchorage of Fap1 on the cell surface is required for long fimbriae formation. These data further document the role of long fimbriae in adhesion of S. parasanguis FW213 to SHA.
Fus3 controls Ty1 transpositional dormancy through the invasive growth MAPK pathway
Authors: Conte Darryl , Curcio M. Joan
Published: March 2003

Fus3, the mitogen‐activated protein kinase (MAPK) of the mating pheromone response pathway, inhibits a post‐translational step of Ty1 retrotransposition. Fus3 also inhibits haploid invasive growth by blocking cross‐activation of invasive growth gene expression by the pheromone response signal cascade. Here, we show that Fus3 kinase activity and dosage co‐ordinately regulate Ty1 transposition and invasive growth. A chromosomal copy of the kinase‐defective fus3‐K42R allele fails to inhibit either Ty1 transposition or invasive growth. When overexpressed, kinase‐defective Fus3 weakly inhibits both Ty1 transposition and invasive growth, but is much less inhibitory than wild‐type Fus3 expressed at the same level. Moreover, increasing the dosage of wild‐type Fus3 intensifies the inhibition of both Ty1 transposition and invasive growth. To demonstrate that Fus3 regulates Ty1 transposition via its negative regulation of the invasive growth pathway, we show by epistatic analysis that the invasive growth pathway transcription factors Ste12 and Tec1 are both required for Fus3‐mediated inhibition of Ty1 transposition. When haploid invasive growth is stimulated by high‐copy expression of TEC1, by expression of the dominant hypermorphic allele STE11‐4 or by deletion of HOG1, Ty1 transposition is concomitantly activated. In summary, these results demonstrate that the haploid invasive growth pathway activates Ty1 transposition at both transcriptional and post‐transcriptional levels and that Fus3 inhibits Ty1 transposition by inhibiting the invasive growth pathway.
Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker
Authors: Peck Ronald F. , DasSarma Shiladitya , Krebs Mark P.
Published: March 2003

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine‐5′‐monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5‐fluoroorotic acid (5‐FOA), which can select for mutations in ura3. A spontaneous 5‐FOA‐resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5‐FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Δura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine‐5′‐monophosphate decarboxylase. To demonstrate ura3‐based gene replacement, a Δbop strain was constructed by transforming a Δura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5‐FOA, yielding Δbop recombinants at a frequency of > 10−2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.
The type III protein translocation system of enteropathogenic Escherichia coli involves EspA–EspB protein interactions
Authors: Hartland Elizabeth L. , Daniell Sarah J. , Delahay Robin M. , Neves Bianca C. , Wallis Tim , Shaw Robert K , Hale Christine , Knutton Stuart , Frankel Gad
Published: March 2003

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for ‘attaching and effacing’ (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co‐immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp‐2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.
The NC2 repressor is dispensable in yeast mutated for the Sin4p component of the holoenzyme and plays roles similar to Mot1p in vivo
Authors: Lemaire Marc , Xie Jun , Meisterernst Michael , Collart Martine A.
Published: March 2003

NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone‐fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA‐bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2β activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA‐less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non‐essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.
Molecular structure of bacterial endotoxin (Escherichia coli Re lipopolysaccharide): implications for formation of a novel heterogeneous lattice structure
Authors: Kato Nobuo , Sugiyama Tsuyoshi , Naito Setsuko , Arakawa Yoshichika , Ito Hideo , Kido Nobuo , Ohta Michio , Sasaki Kyoyu
Published: March 2003

Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3‐deoxy‐dmanno‐octulosonic acid (dOclA) dimer and the N‐acetylglucosamine disaccharide of lipid A. X‐ray diffraction reveals that the type of cell is monoclinic (a = 5.53 Å, b = 27.2 Å, c = 6.47 Å , α = 90°, β = 125.8°, γ = 90°). Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b‐axis value, and two types of surface topographies are visualized. One, regarded as the view onto the acyl chain ends, is two‐dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a‐ and c‐axes and the angle of β (lattice I). The other, regarded as the view onto the dOclA dimers, is two‐dimensional arrays of dromedary‐back‐like bodies that constitute the lattice with axes of 9.0 and 10.7 Å and the angle of 65° formed by both axes (lattice II). Based on these results, we present the molecular model of E. coli Re LPS.
Antisense RNA‐dependent transcription termination sites that modulate lysogenic development of satellite phage P4
Authors: Briani Federica , Ghisotti Daniela , Dehò Gianni
Published: March 2003

In the lysogenic state, bacteriophage P4 prevents the expression of its own replication genes, which are encoded in the left operon, through premature transcription termination. The phage factor responsible for efficient termination is a small, untranslated RNA (CI RNA), which acts as an antisense RNA and controls transcription termination by pairing with two complementary sequences (seqA and seqC) located within the leader region of the left operon. A Rho‐dependent termination site, timm, was previously shown to be involved in the control of P4 replication gene expression. In the present study, by making use of phage ΦR73 as a cloning vector and of suppressor tRNAGly as a reporter gene, we characterized two additional terminators, t1 and t4. Although transcription termination at neither site requires the Rho factor, only t1 has the typical structure of a Rho‐independent terminator. t1 is located between the PLE promoter and the cI gene, whereas t4 is located between cI and timm. Efficient termination at t1 requires the CI RNA and the seqA target sequence; in vitro, the CI RNA enhanced termination at t1 in the absence of any bacterial factor. A P4 mutant, in which the t1 terminator has been deleted, can still lysogenize both Rho+ and Rho− strains and exhibits increased expression of CI RNA. These data indicate that t1 and the Rho‐dependent timm terminators are not essential for lysogeny. t1 is involved in CI RNA autoregulation, whereas t4 appears to be the main terminator necessary to prevent expression of the lytic genes in the lysogenic state.
BofC negatively regulates SpoIVB‐mediated signalling in the Bacillus subtilisσK‐checkpoint
Authors: Wakeley Phil , Hoa Ngo Thi , Cutting Simon
Published: March 2003

The BofC protein acts negatively on intercompartmental signalling of pro‐σK processing in the σK‐checkpoint of Bacillus subtilis. Signalling is brought about by the SpoIVB protein, which is synthesized in the forespore and initiates proteolytic processing of pro‐σK to its mature and active form in the opposed mother cell chamber of the developing cell. We have shown here that BofC, like SpoIVB, is secreted across the inner forespore membrane and, from the analysis of a bofC deletion and insertion mutant, is likely to interact with SpoIVB. In the absence of BofC, the amount of SpoIVB found in sporulating cells is substantially reduced, although SpoIVB is still able to activate proteolysis of pro‐σK. Conversely, in the absence of SpoIVB, the levels of BofC accumulate suggesting that the fate of each molecule is dependent upon their mutual interaction. Our results suggest that BofC could maintain SpoIVB in a stable but inactive form. Supporting this, we have shown that overproduction of BofC inhibits SpoIVB autoproteolysis and leads to a delay in proteolytic cleavage of pro‐σK. Based on our work here, we have proposed a model for BofC′s functional role in intercompartmental signalling.
Membrane topology of the Mep/Amt family of ammonium transporters
Authors: Thomas Gavin H. , Mullins Jonathan G. L. , Merrick Mike
Published: March 2003

The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high‐affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB–PhoA fusion proteins with a complementary set of AmtB–LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane‐spanning helices, with the N‐terminus on the exterior face of the membrane and the C‐terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane‐spanning region at the N‐terminus. Addition of PhoA or LacZ α‐peptide to the C‐terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]‐methylammonium uptake. This C‐terminal region, along with four membrane‐spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp‐182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.
Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle
Authors: Shaw E. I. , Dooley C. A. , Fischer E. R. , Scidmore M. A. , Fields K. A. , Hackstadt T.
Published: March 2003

The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs differentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until released from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial developmental cycle, we have used quantitative–competitive polymerase chain reaction (QC‐PCR) and reverse transcription (RT)‐PCR techniques. These analyses demonstrate that C. trachomatis double their DNA content every 2–3 h, with synthesis beginning between 2 and 4 h after infection. We determined the onset of transcription of specific temporal classes of developmentally expressed genes. RT‐PCR analysis was performed on several genes encoding key enzymes or components of essential biochemical pathways and functions. This comparison encompassed approximately 8% of open reading frames on the C. trachomatis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transcript per infected cell is detected, three major temporal classes of gene expression were resolved. Initiation of transcription appears to occur in three temporal classes which we have operationally defined as: early, which are detected by 2 h after infection during the germination of EBs to RBs; mid‐cycle, which appear between 6 and 12 h after infection and represent transcripts expressed during the growth and multiplication of RBs; or late, which appear between 12 and 20 h after infection and represent those genes transcribed during the terminal differentiation of RBs to EBs. Collectively, the data suggest that chlamydial early gene functions are weighted toward initiation of macromolecular synthesis and the establishment of their intracellular niche by modification of the inclusion membrane. Surprisingly, representative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10–12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs.
Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis
Authors: Slayden Richard A. , Lee Richard E. , Barry Clifton E.
Published: March 2003

Genetic and biochemical evidence has implicated two different target enzymes for isoniazid (INH) within the unique type II fatty acid synthase (FAS) system involved in the production of mycolic acids. These two components are an enoyl acyl carrier protein (ACP) reductase, InhA, and a β‐ketoacyl‐ACP synthase, KasA. We compared the consequences of INH treatment of Mycobacterium tuberculosis (MTB) with two inhibitors having well‐defined targets: triclosan (TRC), which inhibits InhA; and thiolactomycin (TLM), which inhibits KasA. INH and TLM, but not TRC, upregulate the expression of an operon containing five FAS II components, including kasA and acpM. Although all three compounds inhibit mycolic acid synthesis, treatment with INH and TLM, but not with TRC, results in the accumulation of ACP‐bound lipid precursors to mycolic acids that were 26 carbons long and fully saturated. TLM‐resistant mutants of MTB were more cross‐resistant to INH than TRC‐resistant mutants. Overexpression of KasA conferred more resistance to TLM and INH than to TRC. Overexpression of InhA conferred more resistance to TRC than to INH and TLM. Co‐overexpression of both InhA and KasA resulted in strongly enhanced levels of INH resistance, in addition to cross‐resistance to both TLM and TRC. These results suggest that these components of the FAS II complex are not independently regulated and that alterations in the expression level of InhA affect expression levels of KasA. Nonetheless, INH appeared to resemble TLM more closely in overall mode of action, and KasA levels appeared to be tightly correlated with INH sensitivity.
Identification and characterization of in vivo attenuated mutants of Brucella melitensis
Authors: Lestrate P. , Delrue R. ‐M. , Danese I. , Didembourg C. , Taminiau B. , Mertens P. , De Bolle X. , Tibor A. , Tang C. M. , Letesson J.‐J.
Published: March 2003

Brucella melitensis 16M is a Gram‐negative α2‐proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non‐professional phagocytes. Signature‐tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild‐type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone‐like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.
Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function
Authors: Lin B. , Skidmore J. M. , Bhatt A. , Pfeffer S. M. , Pawloski L. , Maddock J. R.
Published: March 2003

The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP‐binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide‐binding and exchange properties of CgtA are different from those of the well‐characterized Ras‐like GTP‐binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector‐binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant‐GDP and mant‐GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg‐like protein results in the lack of function in vivo.
Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1β and inhibits its PKC‐mediated nucleotide exchange activity in vitro
Authors: Mamoun Choukri Ben , Goldberg Daniel E.
Published: March 2003

The elongation step of protein synthesis involves binding of aminoacyl‐tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl‐tRNA to the P site. The nucleotide exchange factor EF‐1β plays a major role in the regulation of this process by regenerating a GTP‐bound EF‐1α necessary for each elongation cycle. EF‐1β has been shown to be phosphorylated and its phosphorylation is critical for optimal activity. We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum. In the current work, we performed Far‐Western analysis to identify PfPP2C substrates. Several components of the translation and transcription machinery were identified, including translation elongation factor 1‐beta (PfEF‐1β). PfEF‐1β is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity. PKC‐phosphorylated PfEF‐1β is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level. The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.
Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12–FK506 in Cryptococcus neoformans
Authors: Fox Deborah S. , Cruz M. Cristina , Sia Rey A. L. , Ke Hengming , Cox Gary M. , Cardenas Maria E. , Heitman Joseph
Published: March 2003

Calcineurin is a Ca2+–calmodulin‐regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37°C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1‐1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two‐amino‐acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12–FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12–FK506–calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.
Chromosomal organization governs the timing of cell type‐specific gene expression required for spore formation in Bacillus subtilis
Authors: Zupancic Margaret L. , Tran Huong , Hofmeister Antje E. M.
Published: March 2003

During the early stages of spore formation in Bacillus subtilis, asymmetric division precedes chromosome segregation, such that the forespore transiently contains only about one‐third of the genetic material surrounding the origin of replication. Shortly after septum formation, the transcription factor σF initiates forespore‐specific gene expression that is essential for the proteolytic activation of pro‐σE in the neighbouring mother cell. Moving the σF‐dependent spoIIR gene from its original origin‐proximal position to an ectopic origin‐distal site caused a delay in spoIIR transcription, as well as delays and reductions in the proteolytic activation of pro‐σE and σE‐directed gene expression. These defects correlated with the accumulation of disporic sporangia, thus reducing sporulation efficiency in a manner that depended upon the distance that spoIIR had been moved from the origin‐proximal third of the chromosome. A significant proportion of disporic sporangia exhibited σE activity in their central compartment, indicating that delays and reductions in σE activation can lead to the formation of a second septum at the opposite pole. These observations support a model in which chromosomal spoIIR position temporally regulates σE activation, thereby allowing for the rapid establishment of mother cell‐specific gene expression that is essential for efficient spore formation. The implications of these findings for cell type‐specific gene expression during the early stages of spore formation in B. subtilis are discussed.
Identification of a non‐haem catalase in Salmonella and its regulation by RpoS (σS)
Authors: Robbe‐Saule Véronique , Coynault Colette , Ibanez‐Ruiz Magdalena , Hermant Daniel , Norel Françoise
Published: March 2003

We report the identification and functional analysis of katN, a gene encoding a non‐haem catalase of Salmonella enterica serotype Typhimurium. katN, which is not present in Escherichia coli, is located between the yciGFE and yciD E. coli homologues in the Salmonella genome. Its predicted protein product has a molecular weight of 31 826 Da and is similar to the Mn‐catalases of Lactobacillus plantarum and Thermus spp. Its product, KatN, was visualized as a 37 kDa protein in E. coli maxicells. A KatN recombinant protein, containing six histidine residues at its C‐terminus, was purified, and its catalase activity was observed on a non‐denaturing polyacrylamide gel. KatN was also visualized by catalase activity gel staining of bacterial cell extracts. Its expression was shown to be regulated by growth phase and rpoS. Northern blotting indicated that kat forms an operon with the upstream yciGFE genes. A putative rpoS‐regulated promoter was identified upstream of yciG. Southern blotting revealed that katN is conserved within Salmonella serovars. katN homologues were found in Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens. A katN mutation did not appear to affect the hydrogen peroxide (H2O2) response of Salmonella. However, the expression of katN increased the H2O2 resistance of unadapted cells in the exponential phase and of rpoS mutants in stationary phase. Thus, KatN may contribute to hydrogen peroxide resistance in Salmonella in certain environmental conditions.
Non‐heritable change of a spirochaete's phenotype by decoration of the cell surface with exogenous lipoproteins
Authors: Bunikis Jonas , Mirian Hossein , Bunikiene Elena , Barbour Alan G.
Published: March 2003

Genetic transformation of Borrelia spp. is limited in development and has found application in only one species. For a non‐genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane. Using unlabelled or 125I‐labelled Osp proteins, Osp‐specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics. (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B. hermsii cells that lacked abundant lipoproteins of their own. (ii) This decoration of the cells with exogenous OspA did not affect cell's viability. (iii) The decoration was concentration and temperature dependent and stable for at least 24 h. (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross‐linked to the integral outer membrane protein, P66. (v) Decoration of viable B. burgdorferi and B. hermsii with OspA rendered the cells susceptible to killing by OspA‐specific antiserum. Such non‐genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ.
Understanding the enzymology of archaeal DNA replication: progress in form and function
Authors: MacNeill Stuart A.
Published: March 2003

The analysis of completed archaeal genome sequences led to the identification of a set of approximately 10–20 genes whose protein products were inferred to be involved in chromosomal DNA replication. Until recently, however, little was known of the biochemical properties of these proteins. Here, I review recent progress in this area brought about by biochemical and structural analysis. Aside from shedding considerable new light on the molecular machinery of DNA replication in the archaea, the results of these studies also present new opportunities for understanding the molecular events of chromosomal DNA replication in eukaryotic cells.
Transcription of essential cell division genes is linked to chromosome replication in Escherichia coli
Authors: Liu Guowen , Begg Ken , Geddes Auste , Donachie William D.
Published: March 2003

Cell division normally follows the completion of each round of chromosome replication in Escherichia coli. Transcription of the essential cell division genes clustered at the mra region is shown here to depend on continuing chromosomal DNA replication. After chromosome replication was blocked by either nalidixic acid treatment or thymine starvation, the transcription of these cell division genes was repressed significantly. This suggests a way in which cell division is controlled by chromosome replication.
Multiple effects of protein phosphatase 2A on nutrient‐induced signalling in the yeast Saccharomyces cerevisiae‡
Authors: Sugajska Ewa , Swiatek Wojciech , Zabrocki Piotr , Geyskens Ilse , Thevelein Johan M. , Zolnierowicz Stanislaw , Wera Stefaan
Published: March 2003

The trehalose‐degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen‐starved glucose‐repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22‐overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress‐regulated element‐controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient‐induced signal transduction restored glucose‐induced trehalase activation in Pph22‐overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient‐induced signal transduction pathways in the absence of nutrients.
Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover
Authors: Mingorance Jesús , Rueda Sonsoles , Gómez‐Puertas Paulino , Valencia Alfonso , Vicente Miguel
Published: March 2003

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter‐binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol−1 protein monomer. At pH 7.5 and in the presence of Mg2+ and K+, there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.
The Neisseria gonorrhoeae lpxLII gene encodes for a late‐functioning lauroyl acyl transferase, and a null mutation within the gene has a significant effect on the induction of acute inflammatory responses
Authors: Ellis Charles D. , Lindner Buko , Khan C. M. Anjam , Zähringer Ulrich , De Hormaeche Raquel Demarco
Published: March 2003

LPS is a fundamental constituent of the outer membrane of all Gram‐negative bacteria, and the lipid A domain plays a central role in the induction of inflammatory responses. We identified genes of the Neisseria gonorrhoeae lipid A biosynthetic pathway by searching the complete gonococcal genome sequence with sequences of known enzymes from other species. The lpxLII gene was disrupted by an insertion–deletion in an attenuated aroA mutant of the gonococcal strain MS11. Lipopolysaccharide (LPS) and lipid A analysis demonstrated that the lpxLII mutant had synthesized an altered LPS molecule lacking a single lauric fatty acid residue in the GlcN II of the lipid A backbone. LPS of the lpxLII mutant had a markedly reduced ability to induce the proinflammatory cytokines tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 and IL‐8 from human macrophages and IL‐8 from polymorphonuclear cells. This study demonstrates that the lpxLII gene in gonococci encodes for a late‐functioning lauroyl acyl transferase that adds a lauric acid at position 2′ in the lipid A backbone. The presence of lauric acid at such a position appears to be crucial for the induction of full inflammatory responses by N. gonorrhoeae LPS.
Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision
Authors: Hinerfeld Douglas , Churchward Gordon
Published: March 2003

The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.