bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri : characterization of four fnr‐like genes, regulatory responses and cognate metabolic processes
Authors: Vollack Kai‐Uwe , Härtig Elisabeth , Körner Heinz , Zumft Walter G.
Published: March 2003

Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR‐type regulator, which is expressed constitutively and controls the cytochrome cbb3‐type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen‐independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe–4S] centre of redox‐active FNR‐type regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen‐limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcription of dnrD was activated in O2‐limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA. In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD. dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase. The transcription of dnrS required FnrA in O2‐limited cells. Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response. The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction. We found the highest amount of dnrE transcripts in aerobically nitrate‐challenged cells. The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL. The multiplicity of FNR factors in P. stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate‐activated metabolic processes.
Universal replication biases in bacteria
Authors: Rocha Eduardo P. C. , Danchin Antoine , Viari Alain
Published: March 2003

Analysis of 15 complete bacterial chromosomes revealed important biases in gene organization. Strong compositional asymmetries between the genes lying on the leading versus lagging strands were observed at the level of nucleotides, codons and, surprisingly, amino acids. For some species, the bias is so high that the sole knowledge of a protein sequence allows one to predict with almost no errors whether the gene is transcribed from one strand or the other. Furthermore, we show that these biases are not species specific but appear to be universal. These findings may have important consequences in our understanding of fundamental biological processes in bacteria, such as replication fidelity, codon usage in genes and even amino acid usage in proteins.
Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant
Authors: Nishiyama So‐ichiro , Umemura Tohru , Nara Toshifumi , Homma Michio , Kawagishi Ikuro
Published: March 2003

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate‐bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl‐accepting glutamyl residues with non‐methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB‐defective, CheR‐overproducing strain. The fully glutamyl‐methylated proteins were designed to mimic the full range of methylation states possible for wild‐type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature‐dependent signalling properties.
Host‐induced, stage‐specific virulence gene activation in Candida albicans during infection
Authors: Staib Peter , Kretschmar Marianne , Nichterlein Thomas , Köhler Gerwald , Michel Sonja , Hof Herbert , Hacker Jörg , Morschhäuser Joachim
Published: March 2003

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP‐mediated genetic recombination. The FLP gene, encoding the site‐specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P–FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid‐sensitive phenotype of the colonies generated from such cells after FLP‐mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.
The multiply‐regulated gabA gene encoding the GABA permease of Aspergillus nidulans: a score of exons
Authors: Hutchings Helen , Stahmann K.‐Peter , Roels Steven , Espeso Eduardo A. , Timberlake William E. , Arst, Jr Herbert N. , Tilburn Joan
Published: March 2003

We describe the cloning, sequence and expression of gabA, encoding the γ‐amino‐n‐butyrate (GABA) permease of the fungus Aspergillus nidulans. Sequence changes were determined for three up‐promoter (gabI ) and six gabA loss‐of‐function mutations. The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease. Structural predictions favour 11–12 transmembrane domains. Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene. In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, ω‐amino acid induction and regulation in response to ambient pH (being acid‐expressed). In agreement with this, we report consensus binding sites 5′ to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.
The mutA mistranslator tRNA‐induced mutator phenotype requires recA and recB genes, but not the derepression of lexA‐regulated functions
Authors: Ren Li , Al Mamun Abu Amar M. , Humayun M. Zafri
Published: March 2003

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a ΔrecA strain raise the possibility that asp → gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA‐promoted asp → gly misinsertion; or (iii) a hitherto unrecognized recA‐dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC ::lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild‐type lexA allele with the lexA1[Ind‐] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA‐promoted asp → gly misinsertion occurs in recA− cells and, therefore, the requirement for recA is ‘downstream’ of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.
Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays
Authors: Gordon Stephen V. , Brosch Roland , Billault Alain , Garnier Thierry , Eiglmeier Karin , Cole Stewart T.
Published: March 2003

Whole‐genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG‐Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction‐digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1–RD10) relative to M. tuberculosis. Seven of these regions, RD4–RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG‐specific deletions being identical to the RD1–RD3 loci described previously. The distribution of RD4–RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT‐6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.
Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation
Authors: Raynal Lelia C. , Carpousis Agamemnon J.
Published: March 2003

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA‐adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer‐independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North‐western and Far‐western blotting using truncated forms of PAP I. Additional protein–protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N‐terminal catalytic domain, a C‐terminal RNA binding domain and sites for the interaction with other protein factors.
Multiple lysophosphatidic acid acyltransferases in Neisseria meningitidis
Authors: Shih Giles C. , Kahler Charlene M. , Swartley John S. , Rahman M. Mahbubur , Coleman Jack , Carlson Russell W. , Stephens David S.
Published: March 2003

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are critical phospholipid intermediates in the biosynthesis of cell membranes. In Escherichia coli, LPA acyltransferase (1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase; EC 2.3.1.51) catalyses the transfer of an acyl chain from either acyl‐coenzyme A or acyl–acyl carrier protein onto LPA to produce PA. While E. coli possesses one essential LPA acyltransferase (PlsC), Neisseria meningitidis possesses at least two LPA acyltransferases. This study describes the identification and characterization of nlaB (neisserial LPA acyltransferase B), the second LPA acyltransferase identified in N. meningitidis. The gene was located downstream of the Tn916 insertion in N. meningitidis mutant 469 and differed in nucleotide and predicted amino acid sequence from the previously characterized neisserial LPA acyltransferase homologue nlaA. NlaB has specific LPA acyltransferase activity, as demonstrated by complementation of an E. coli plsC(Ts) mutant in trans, by decreased levels of LPA acyltransferase activity in nlaB mutants and by lack of complementation of E. coli plsB26,X50, a mutant defective in the first acyltransferase step in phospholipid biosynthesis. Meningococcal nlaA mutants accumulated LPA and demonstrated alterations in membrane phospholipid composition, yet retained LPA acyltransferase activity. In contrast, meningococcal nlaB mutants exhibited decreased LPA acyltransferase activity, but did not accumulate LPA or display any other observable membrane changes. We propose that N. meningitidis possesses at least two LPA acyltransferases to provide for the production of a greater diversity of membrane phospholipids.
Phase variation of HpuAB and HmbR, two distinct haemoglobin receptors of Neisseria meningitidis DNM2
Authors: Lewis L. A. , Gipson M. , Hartman K. , Ownbey T. , Vaughn J. , Dyer D. W.
Published: March 2003

We have previously described HpuAB, a two‐component receptor that mediates binding to haemoglobin (Hb), haemoglobin–haptoglobin (Hb‐Hp) and apo‐haptoglobin (Hp). In this communication, we constructed non‐polar mutations in the hpuA and hpuB loci to examine the individual roles of HpuA and HpuB. Our results indicate that both HpuA and HpuB are required for the acquisition of Fe from Hb and Hb‐Hp. We isolated Hb utilization‐positive (Hb+) variants of our Hb utilization‐negative (Hb−) hpu mutants at a frequency of 10−3 and demonstrated that the Hb+ phenotype resulted from the expression of a second Hb receptor, HmbR. Expression of HmbR in DNM2 was found to be controlled by translational frameshifting involving a polyguanine (G) tract located within the hmbR locus. The hpuA locus also contains a poly(G) tract, which suggested that meningococci could phase vary each Hb receptor independently by slip‐strand mispairing in the poly(G) tracts found in hpuA and hmbR. Thus, we isolated a naturally occurring Hb− variant of DNM2, designated DNM2 Hb−, which did not express either HpuAB or HmbR. Hb+ variants of DNM2Hb− were selected and examined for HpuAB and HmbR expression. In each instance, acquisition of HpuAB or HmbR expression was correlated with phase variation in the poly(G) tract of each Hb receptor.
Post‐transcriptional regulation of the Bacillus subtilis dnaK operon
Authors: Homuth Georg , Mogk Axel , Schumann Wolfgang
Published: March 2003

The heptacistronic dnaK heat shock operon of Bacillus subtilis consists of the genes hrcA, grpE, dnaK, dnaJ, orf35, orf28 and orf50. It is controlled by the CIRCE/HrcA operator/repressor system and specifies three primary transcripts, two of which are processed into three different products. We have analysed the regulatory consequences of this complex transcriptional organization in detail. First, the seven genes were heat induced to different extents at the mRNA level and can be classified into three groups by their induction factors. This differential induction was also reflected at the protein level. Secondly, the cellular amounts of the proteins HrcA, DnaK and DnaJ in B. subtilis differed drastically both under non‐heat shock conditions and after thermal upshock. Thirdly, Northern blot analyses demonstrated that an mRNA‐processing reaction generating products of differential stabilities plays an essential role during the regulation of gene expression. A crucial factor determining the low stability of two transcripts is the presence of the CIRCE element at their 5′ ends. We demonstrate that CIRCE leads to the destabilization of mRNAs, but only if it is located in the immediate vicinity of a Shine–Dalgarno sequence. These results show that B. subtilis is using various, especially post‐transcriptional, regulatory mechanisms to fine tune the expression of the individual genes of the heptacistronic dnaK operon.
Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis
Authors: Lavitola Alfredo , Bucci Cecilia , Salvatore Paola , Maresca Gabriella , Bruni Carmelo B. , Alifano Pietro
Published: March 2003

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/−1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho‐dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re‐expression (off–on) without affecting the rates of loss of capsule expression (on–off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD–ermC′ gene fusions in wild type and Rho‐defective Escherichia coli strains. These studies demonstrated that in the Rho‐defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off–on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD–ermC′ gene fusions in a DNA mismatch‐defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.
Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli
Authors: Chang Ying‐Ying , Cronan John E.
Published: March 2003

Cyclopropane fatty acid (CFA) formation is a post‐synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid. The synthesis of CFA depends in part on the RpoS sigma factor, but the role of RpoS in resistance to acid shock involves additional factors because strains with null mutations in both cfa and rpoS are more sensitive to acid shock than either single mutant strain. Exponential phase cultures of E. coli are much more sensitive to acid shock than stationary phase cultures, but survival is greatly increased if the exponential phase cultures are exposed to moderately acid conditions (pH 5) before shift to pH 3. We show that exposure to moderately acid conditions gives a marked increase in cfa transcription. The efficiency of the survival of acid shock is extremely strain dependent, even among putative wild‐type strains. Much, but not all, of this variability can be explained by the partially or totally defective RpoS alleles carried by many strains.
Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli
Authors: Martínez‐Laguna Ygnacio , Calva Edmundo , Puente José Luis
Published: March 2003

Expression of bfpA, the gene coding for the structural subunit of the bundle‐forming pili (BFP) in enteropathogenic Escherichia coli (EPEC), requires the product of bfpT (also called perA), a member of the AraC family of transcriptional regulators. Here, we show that bfpT–cat fusions were not expressed in a bfpT − or in a non‐EPEC strain, unless a functional bfpT was present, indicating that an autoregulatory mechanism is involved in expression. Further experiments with bfpT–cat fusions and primer extension analysis showed that bfpT is transcribed from a conventional sigma‐70 promoter and that it is expressed throughout the growth curve. It is regulated in response to the ammonium concentration, temperature and growth media, in the same proportions as those described previously for bfpA. In addition, bfpT and bfpA expression was also modulated by osmolarity, but was not affected by pH, iron excess or limitation. Deletion analysis of the bfpT upstream region revealed that a DNA segment of 81 bp, extending upstream from the transcriptional start site, contained all the sequence elements required for maximal expression of bfpT. Furthermore, it shares significant homology with a bfpA upstream AT‐rich region, which has been shown to be involved in the BfpT‐dependent regulation of bfpA. Interestingly, ammonium repression was observed only when bfpT–cat or bfpA–cat expression was complemented in an EPEC background, whereas low‐temperature regulation was observed in both EPEC and non‐EPEC strains. This suggests that specific regulatory elements are present in EPEC, while others are shared with non‐pathogenic E. coli.
Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis
Authors: Niederweis Michael , Ehrt Sabine , Heinz Christian , Klöcker Uta , Karosi Stefanie , Swiderek Kristine M. , Riley Lee W. , Benz Roland
Published: March 2003

Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel‐forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N‐terminal signal sequence. MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel‐forming activity as whole‐cell extracts of M. smegmatis with organic solvents. These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel‐forming activity. Database searches did not reveal significant similarities to any other known protein. Southern blots showed that the chromosomes of fast‐growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel‐forming proteins.
Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation
Authors: Jiang Min , Tzeng Yih‐Ling , Feher Victoria A. , Perego Marta , Hoch James A.
Published: March 2003

Five single alanine substitution mutations in the Spo0F response regulator gave rise to mutant strains of Bacillus subtilis with seemingly normal sporulation that nevertheless rapidly segregated variants blocked in sporulation. The basis for this deregulated phenotype was postulated to be increased phosphorylation of the Spo0A transcription factor, resulting from enhanced phosphate input or decreased dephosphorylation of the phosphorelay. Strains bearing two of these Spo0F mutant proteins, Y13A and I17A, retained a requirement for KinA and KinB kinases in sporulation, whereas the remaining three, L66A, I90A and H101A, gave strains that sporulated well in the absence of both KinA and KinB. Sporulation of strains bearing L66A and H101A mutations was decreased in a mutant lacking KinA, KinB and KinC, but the strain bearing the I90A mutation required the further deletion of KinD to lower its sporulation frequency. The affected residues, L‐66, I‐90 and H‐101, are involved in crucial hydrophobic contacts stabilizing the orientation of helix α4 of Spo0F. The data are consistent with the notion that these three mutations alter the conformation of the β4–α4 loop of Spo0F that is known to contain residues critical for KinA:Spo0F recognition. As this loop has a propensity for multiple conformations, the spatial arrangement of this loop may play a critical role in kinase selection by Spo0F and might be altered by regulatory molecules interacting with Spo0F.
The integrase family of recombinases: organization and function of the active site
Authors: Grainge Ian , Jayaram Makkuni
Published: March 2003

The integrase family of site‐specific recombinases (also called the tyrosine recombinases) mediate a wide range of biological outcomes by the sequential exchange of two pairs of DNA strands at defined phosphodiester positions. The reaction produces a recombinant arrangement of the DNA sequences flanking the cross‐over region. The crystal structures of four integrase family members have revealed very similar three‐dimensional protein folds that belie the large diversity in amino acid sequences among them. The active sites are similar in organization to those seen in structures of eukaryotic type IB topoisomerases, and conservation of catalytic mechanism is expected. The crystal structures, combined with previous biochemical knowledge, allow the refinement of models for recombination and the assignment of catalytic function to the active site residues. However, each system has its own peculiarities, and the exact sequence of events that allows the reaction to proceed from the first exchange reaction to the second is still unclear for at least some family members.
The opcA and ΨopcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands
Authors: Zhu Peixuan , Morelli Giovanna , Achtman Mark
Published: March 2003

Previous data have indicated that the opc gene encoding an immunogenic invasin is specific to Neisseria meningitidis (Nm) and is lacking in Neisseria gonorrhoeae (Ng). The data presented here show that Nm and Ng both contain two paralogous opc‐like genes, opcA, corresponding to the former opc gene, and ΨopcB, a pseudogene. The predicted OpcA and OpcB proteins possess transmembrane regions with conserved non‐polar faces but differ extensively in four of the five surface‐exposed loops. Gonococcal OpcA was expressed weakly under in vitro conditions, and it is unknown whether these bacteria can express this protein at high levels. Analysis of the sequences flanking opcA and ΨopcB revealed a framework of conserved housekeeping genes interspersed with DNA islands. These regions also contained several pseudogenes, deletions and IS elements, attesting to considerable genome plasticity. Both opcA and ΨopcB are located on DNA islands that have probably been imported from unrelated bacteria. A third island encodes the dcmD/dcrD R/M genes in Ng versus a small open reading frame in most strains of Nm. Rare strains of Nm were identified in which the R/M island has been imported. DNA islands in Nm and Ng seem to have been acquired by recombination via conserved flanking housekeeping genes rather than by insertion of mobile genetic elements.
Resistance mutations reveal the atovaquone‐binding domain of cytochrome b in malaria parasites
Authors: Srivastava Indresh K. , Morrisey Joanne M. , Darrouzet Elisabeth , Daldal Fevzi , Vaidya Akhil B.
Published: March 2003

Atovaquone represents a class of antimicrobial agents with a broad‐spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria‐containing parasites. To that end, we derived nine independent atovaquone‐resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone‐mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance‐conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side‐chains may determine atovaquone‐binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.
Induction of the mtrCDE‐encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an AraC‐like protein
Authors: Rouquette Corinne , Harmon Jennifer B. , Shafer William M.
Published: March 2003

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy‐dependent efflux pump composed of the MtrC–MtrD–MtrE cell envelope proteins that serves to export structurally diverse antimicrobial, hydrophobic agents (HAs). Many of these agents have membrane‐acting detergent activity. Using Triton X‐100 (TX‐100) as a representative HA, we found that the mtrCDE efflux pump operon could be induced to higher levels of expression when an HA‐sensitive strain was exposed to sublethal concentrations of this non‐ionic detergent and the structurally related spermicide, nonoxynol‐9. This induction was at the level of mtrCDE gene transcription and was independent of the MtrR repressor, which normally decreases mtrCDE gene expression. However, the enhanced resistance of gonococci to TX‐100 was dependent on the expression of a previously undescribed gonococcal protein that belonged to the AraC/XylS family of transcriptional activators. We have termed this protein MtrA to signify its likely role in the activation of mtrCDE gene expression. Taken together with previous studies dealing with the genetic control of mtrCDE gene expression, we propose that gonococci can modulate their resistance to HAs through both positive and negative transcriptional control processes. The action of these regulatory processes is probably of importance in determining the survival capacity of gonococci at mucosal surfaces that contain detergent‐like HAs.
Pseudomonas aeruginosa AlgZ, a ribbon–helix–helix DNA‐binding protein, is essential for alginate synthesis and algD transcriptional activation
Authors: Baynham Patricia J. , Brown Amy L. , Hall Lisa L. , Wozniak Daniel J.
Published: March 2003

The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high‐level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS–PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130‐fold enrichment for AlgZ, was devised, the protein identified and a partial amino‐terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon–helix–helix family of DNA‐binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non‐mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.
The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence
Authors: Vasil Michael L. , Ochsner Urs A.
Published: March 2003

During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator — Fur) and a Fur‐regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.
A system for identifying post‐invasion functions of invasion genes: requirements for the Mxi–Spa type III secretion pathway of Shigella flexneri in intercellular dissemination
Authors: Schuch Raymond , Sandlin Robin C. , Maurelli Anthony T.
Published: March 2003

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi–Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin‐based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell‐to‐cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post‐invasion‐specific processes, distinct from its previously characterized roles in invasion. Studies of post‐invasion Ipa and Mxi–Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long‐term intracellular proliferation and viability. Examination of TIER mutant‐infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread — bacterial escape from protrusion‐derived, double‐membrane‐bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi–Spa is required in the protrusion vacuole for double‐membrane lysis.
Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite‐resistant Leishmania
Authors: Haimeur Anass , Guimond Chantal , Pilote Sylvie , Mukhopadhyay Rita , Rosen Barry P. , Poulin Richard , Ouellette Marc
Published: March 2003

The levels of trypanothione, a glutathione–spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate‐limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene amplification in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene amplification. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is amplified in the mutants. The co‐transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.
Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila
Authors: Segal Gil , Russo James J. , Shuman Howard A.
Published: March 2003

We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL‐60‐derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.
A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli
Authors: Schneider Robert , Travers Andrew , Kutateladze Tamara , Muskhelishvili Georgi
Published: March 2003

In Escherichia coli, the transcriptional activity of many promoters is strongly dependent on the negative superhelical density of chromosomal DNA. This, in turn, varies with the growth phase, and is correlated with the overall activity of DNA gyrase, the major topoisomerase involved in the elevation of negative superhelicity. The DNA architectural protein FIS is a regulator of the metabolic reorganization of the cell during early exponential growth phase. We have previously shown that FIS modulates the superhelical density of plasmid DNA in vivo, and on binding reshapes the supercoiled DNA in vitro. Here, we show that, in addition, FIS represses the gyrA and gyrB promoters and reduces DNA gyrase activity. Our results indicate that FIS determines DNA topology both by regulation of topoisomerase activity and, as previously inferred, by directly reshaping DNA. We propose that FIS is involved in coupling cellular physiology to the topology of the bacterial chromosome.
The MAP kinase Kpp2 regulates mating and pathogenic development in Ustilago maydis
Authors: Müller Philip , Aichinger Christian , Feldbrügge Michael , Kahmann Regine
Published: March 2003

In the phytopathogenic fungus Ustilago maydis, fusion of compatible haploid cells is a prerequisite for infection. This process is genetically controlled by the biallelic a locus, encoding pheromone precursors and receptors. These are presumed to be coupled to a heterotrimeric G protein and a MAP kinase cascade, leading to activation of the HMG domain transcription factor Prf1. Here, we have demonstrated that putative MAP kinase sites in Prf1 are required for its activity during mating. In addition, we have identified a gene, kpp2, which encodes a putative MAP kinase related to Pmk1 of Magnaporthe grisea and Fus3p of Saccharomyces cerevisiae. kpp2 deletion mutants are attenuated in several steps of development: cell fusion, induction of pheromone‐responsive genes and pathogenicity. Epistasis analysis shows that kpp2 does not affect pheromone gene expression through the cAMP signalling cascade. Pathogenicity of kpp2 mutants can be partially restored by overexpressing the b genes, indicating a regulation of Prf1 by Kpp2. These data support the hypothesis that the MAP kinase Kpp2 transmits the pheromone signal.
The NADH oxidase of Streptococcus pneumoniae : its involvement in competence and virulence
Authors: Auzat Isabelle , Chapuy‐Regaud Sabine , Le Bras Gisèle , Dos Santos Delphine , Ogunniyi A. David , Le Thomas Isabelle , Garel Jean‐Renaud , Paton James C. , Trombe Marie‐Claude
Published: March 2003

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase‐deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence‐regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.