bluuebunny/crossref_metadata_2025 huggingface dataset:
The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague
Authors: Bearden Scott W. , Perry Robert D.
Published: March 2003

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an ≈5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron‐deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm−) strains, comprises five genes arranged in two distinct operons (yfeA–D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm−, Yfe− (ΔyfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2′‐dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt−, ΔyfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 × 107 cfu) compared with its parental ybt−, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, ΔyfeAB mutant of Y. pestis had an ≈100‐fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.
Repression at a distance by the global regulator KorB of promiscuous IncP plasmids
Authors: Jagura‐Burdzy Grazyna , Macartney Donia P. , Zatyka Malgorzata , Cunliffe Lesley , Cooke Dunstan , Huggins Cerys , Westblade Lars , Khanim Farhat , Thomas Christopher M.
Published: March 2003

KorB protein (358 amino acids) binds to 12 highly conserved sequences on the RK2 genome and co‐ordinates the expression of at least five operons encoding genes for stable inheritance and plasmid transfer. KorB represses the trfA, korA and klaA promoters where it binds 4 bp upstream of the −35 region (class I KorB operators, OB). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where OB is between 80 and 189 bp away from the transcription start point (class II operator). A C‐terminal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His6‐tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing OB sequences. At the trbBp region, where OB9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp−35 region had no effect on the ability of KorB to repress trbBp. Nevertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of OB9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA polymerase may interact directly with KorB bound at OB9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.
MIC231, a naturally occurring mobile insertion cassette from Bacillus cereus
Authors: Chen Yahua , Braathen Pia , Léonard Catherine , Mahillon Jacques
Published: March 2003

Recent dissection of numerous plasmids and transposable elements has given more credence to the modular organization of these genetic and genomic entities. Although many variations on each theme exist, the number of basic functional cassettes is thought to be relatively limited. In this paper, a novel type of mobile cassette is described. A naturally occurring assemblage consisting of two left IS231 ends flanking a d‐stereospecific endopeptidase (adp ) gene was found in several natural isolates of Bacillus cereus. This 1.9 kb genetic entity was shown to transpose in the presence of IS231A transposase, not only in Escherichia coli but also in Bacillus. The acronym MIC231 is proposed for this mobile insertion cassette trans‐activated (teletransposed) by IS231. Using (d‐Phe)4 tetrapeptide as substrate, the endopeptidase activity of the MIC231 adp gene could be demonstrated in E. coli and B. subtilis. Interestingly, this d‐stereospecific endopeptidase activity was not limited to the original B. cereus isolates but was also detected in all but one of the 69 B. cereus sensu lato strains tested, indicating its important, yet dispensable, biological function. However, inactivation of the MIC231 adp gene in two B. cereus strains did not result in any detectable variation of their activity on (d‐Phe)4, suggesting the presence of other distantly related adp gene(s). Thus, although the exact role of MIC231 adp remains elusive, its presence inside a mobile cassette represents the archetype of a novel insertion sequence modular organization.
Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP‐binding cassette mutations on the assembly and function of the T‐DNA transporter
Authors: Dang Tu Anh , Zhou Xue‐Rong , Graf Brenda , Christie Peter J.
Published: March 2003

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T‐DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide‐binding motif are non‐functional and exert a dominant negative phenotype when synthesized in wild‐type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino‐terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to λ phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent‐resistant complex of native protein and a functional, histidine‐tagged derivative by precipitation with anti‐His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His‐VirB4 in A. tumefaciens, indicating that ATP binding is not required for self‐association. A dimerization domain was localized to a proposed N‐terminal membrane‐spanning region of VirB4, as shown by the dominance of an allele coding for the N‐terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB‐dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4′312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP‐dependent activity is required for configuring this channel as a dedicated export machine.
Interactions of HasA, a bacterial haemophore, with haemoglobin and with its outer membrane receptor HasR
Authors: Létoffé Sylvie , Nato Faridabano , Goldberg Michel E. , Wandersman Cécile
Published: March 2003

The major mechanism by which bacteria acquire free or haemoglobin‐bound haem involves direct binding of haem to specific outer membrane receptors. Serratia marcescens and Pseudomonas aeruginosa have an alternative system, which involves an extracellular haemophore, HasA, that captures free or haemoglobin‐bound haem and shuttles it to a specific cell surface outer membrane receptor, HasR. Both haem‐free (apoprotein) and haem‐loaded (holoprotein) HasA bind to HasR, evidence for direct protein–protein interactions between HasA and HasR. HasA binding to HasR takes place in a tonB mutant. TonB is thus required for a step subsequent to HasA binding.
PDZ domains determine the native oligomeric structure of the DegP (HtrA) protease
Authors: Sassoon Nathalie , Arié Jean‐Philippe , Betton Jean‐Michel
Published: March 2003

DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures. Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer. To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains. This truncated variant, DegPΔ, exhibited no proteolytic activity but exerted a dominant‐negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP. Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP.
A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT‐regulated chaperone‐like protein, TrcA, involved in microcolony formation by EPEC
Authors: Tobe Toru , Tatsuno Ichiro , Katayama Eisaku , Wu Cheng‐Yen , Schoolnik Gary K. , Sasakawa Chihiro
Published: March 2003

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle‐forming pili (BFP). An immobilized T7‐tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to ‘fish out’ from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW‐dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non‐virulent E. coli laboratory strains, DH5α and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW‐activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1–His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone‐like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT‐regulated chaperone‐like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.
Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene
Authors: Brown Jr Douglas H. , Giusani Angela D. , Chen Xi , Kumamoto Carol A.
Published: March 2003

Hyphal growth in the opportunistic fungal pathogen Candida albicans is believed to contribute to the virulence of the organism by promoting penetration of fungal cells into host tissue. In this study, stimulation of hyphal growth by a feature of the physical environment was demonstrated. Specifically, growth of cells embedded within a matrix promoted the formation of hyphae. The CZF1 gene, encoding a putative transcription factor, was shown to be involved in the regulation of hyphal growth under certain conditions, including embedded conditions. Ectopic expression of CZF1 in embedded cells promoted the rapid formation of hyphae. Elimination of CZF1 and CPH1, encoding a homologue of the Saccharomyces cerevisiae Ste12p transcription factor, led to a pronounced defect in filamentous growth of embedded cells. Elimination of CZF1 alone led to a moderate defect in hyphal growth under some conditions, including embedded conditions. Hyphal morphogenesis in response to matrix embedding may occur in the opportunistic pathogen, C. albicans, to promote invasion of fungal cells into host tissue.
The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori
Authors: Spohn Gunther , Scarlato Vincenzo
Published: March 2003

In the present study, we provide evidence that the groESL, hrcA–grpE–dnaK and cbpA–hspR–orf operons encoding the major chaperones of the human gastric pathogen Helicobacter pylori are transcribed by the vegetative sigma factor σ80 and are regulated negatively by the transcriptional repressor HspR. In vitro studies with purified recombinant HspR protein established that the protein represses transcription by binding to large DNA regions centred around the transcription initiation site in the case of the Pcbp promoter, and around −85 and −120 in the case of the Pgro and Phrc promoters respectively. All three binding sites contain DNA motifs with some similarity to the HAIR sequence identified as a consensus for the HspR protein of Streptomyces. In contrast to the situation in Streptomyces, in which transcription of HspR‐regulated genes is induced in response to heat shock, transcription of the HspR‐dependent genes in H. pylori is not inducible by thermal stimuli. Transcription of the groESL and cbpA–hspR–orf operons is induced by osmotic shock, while transcription of the hrcA–grpE–dnaK operon, although HspR dependent, is not affected by salt treatment. The possibility that HspR could constitute a global transcriptional regulator for diverse cellular functions with implications for pathogenesis is discussed.
Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser‐56
Authors: Appleby Jeryl L. , Bourret Robert B.
Published: March 2003

The site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp‐57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser→Ala substitution at this position in wild‐type CheY had no effect on function. However, replacement of Ser‐56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser‐56 or Asp‐57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid–base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu‐57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.
Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli
Authors: Takata Makoto , Guo Lei , Katayama Tsutomu , Hase Masakazu , Seyama Yousuke , Miki Takeyoshi , Sekimizu Kazuhisa
Published: March 2003

We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20°C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30°C, all three mutant proteins exhibited specific activity similar to that seen with the wild‐type protein, whereas at 20°C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild‐type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild‐type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold‐sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.
Interactions between Pho85 cyclin‐dependent kinase complexes and the Swi5 transcription factor in budding yeast
Authors: Measday Vivien , McBride Helen , Moffat Jason , Stillman David , Andrews Brenda
Published: March 2003

Pho85 is a cyclin‐dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28‐dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2‐Pho85, we performed a two‐hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2‐interacting protein. We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two‐hybrid assay. We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80‐Pho85 kinase. We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Δ pho85Δ double mutant. We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain. Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin‐dependent kinase complexes.
Involvement of differential efficiency of transcription by Eσs and Eσ70 RNA polymerase holoenzymes in growth phase regulation of the Escherichia coli osmE promoter
Authors: Bordes Patricia , Repoila Francis , Kolb Annie , Gutierrez Claude
Published: March 2003

Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth. osmE expression is directed by a single promoter, osmEp. Decelerating phase induction of osmEp is dependent on the σs (RpoS) factor, whereas its osmotic induction is independent of σs. Purified Eσs and Eσ70 were both able to transcribe osmEpin vitro on supercoiled templates. In the presence of rpoD800, a mutation resulting in a thermosensitive σ70 factor, a shift to non‐permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting osmEp activity were isolated. Down‐promoter mutations decreased transcription in both the presence and the absence of σs, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter. Three up‐promoter mutations brought osmEp closer to the consensus of Eσ70‐dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of σsin vivo. Our data suggest that Eσs transcribes wild‐type osmEp with a higher efficiency than Eσ70. A model in which an intrinsic differential recognition contributes to growth phase‐dependent regulation is proposed. Generalization of this model to other σs‐dependent promoters is discussed.
Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, Topoisomerase I and Fis
Authors: Weinstein‐Fischer Dalit , Elgrably‐weiss Maya , Altuvia Shoshy
Published: March 2003

Bacterial cells respond to the deleterious effects of reactive oxygen species by inducing the expression of antioxidant defence genes. Here we show that treatment with hydrogen peroxide leads to a transient decrease in DNA negative supercoiling. We also report that hydrogen peroxide activates topA P1 promoter expression. The peroxide‐dependent topA P1 activation is independent of oxyR, but is mediated by Fis. This nucleoid‐associated protein binds to the promoter region of topA. We also show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide. Our results suggest that topA activation by Fis is an important component of the Escherichia coli response to oxidative stress.
Intrinsic instability of the essential cell division protein FtsL of Bacillus subtilis and a role for DivIB protein in FtsL turnover
Authors: Daniel Richard A. , Errington Jeff
Published: March 2003

Cell division in most eubacteria is driven by an assembly of about eight conserved division proteins. These proteins form a ring structure that constricts in parallel with the formation of the division septum. Here, we show that one of the division proteins, FtsL, is highly unstable. We also show that the protein is targeted to the ring structure and that targeting occurs in concert with the recruitment of several other membrane‐associated division proteins. FtsL stability is further reduced in the absence of DivIB protein (probably homologous to E. coli FtsQ) at high temperature, suggesting that DivIB is involved in the control of FtsL turnover. The reduced stability of FtsL may explain the temperature dependence of divIB mutants, because their phenotype can be suppressed by overexpression of FtsL. The results provide new insights into the roles of the FtsL and DivIB proteins in bacterial cell division.
Variable human minisatellite‐like regions in the Mycobacterium tuberculosis genome
Authors: Supply Philip , Mazars Edith , Lesjean Sarah , Vincent Véronique , Gicquel Brigitte , Locht Camille
Published: March 2003

Mycobacterial interspersed repetitive units (MIRUs) are 40–100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite‐like structures evolve slowly in mycobacterial populations.
Interactions of Neisseria meningitidis with cells of the human meninges
Authors: Hardy Samantha J. , Christodoulides Myron , Weller Roy O. , Heckels John E.
Published: March 2003

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low‐adhesive pili, but did not influence the association of high‐adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.
Regulation of intron function: efficient splicing in vivo of a bacterial group II intron requires a functional promoter within the intron
Authors: Zhou Liang , Manias Dawn A. , Dunny Gary M.
Published: March 2003

Conjugative transfer of the Lactococcus lactis plasmid pRS01 requires splicing of a group II intron, Ll.ltrB, for accurate translation of the mRNA for the exon gene ltrB. The protein product of ltrB is a conjugative relaxase, essential for pRS01 transfer. Using a molecular technique for the identification of transcription initiation sites in bacteria, a functional promoter within Ll.ltrB was identified upstream from the gene for the intron‐encoded protein (IEP) LtrA. LtrA is required for efficient splicing of Ll.ltrB in vivo. Mutation of the ltrA promoter dramatically reduced the steady‐state level of ltrA mRNA, LtrA, intron splicing and conjugation in L. lactis. These effects could be relieved by expression in trans of the ltrA gene cloned under the control of an inducible promoter. These results suggest that the ltrA mRNAs are translated inefficiently. We hypothesize that this bacterial intron, in contrast to previously studied group II introns in eukaryotes, requires a promoter within the intron to regulate ltrA expression and to produce an adequate level of the protein in the cell for efficient splicing.
Molecular and physiological analysis of an OxyR‐regulated ahpC promoter in Xanthomonas campestris pv. phaseoli
Authors: Loprasert Suvit , Fuangthong Mayuree , Whangsuk Wirongrong , Atichartpongkul Sopapan , Mongkolsuk Skorn
Published: March 2003

In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR‐regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the −35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the −35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress.
The dppA gene of Bacillus subtilis encodes a new d‐aminopeptidase
Authors: Cheggour Abdelatif , Fanuel Laurence , Duez Colette , Joris Bernard , Bouillenne Fabrice , Devreese Bart , Van Driessche Gonzales , Van Beeumen Jozef , Frère Jean‐Marie , Goffin Colette
Published: March 2003

Different strains of Bacillus were screened for their ability to hydrolyse d‐alanyl‐p‐nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N‐terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non‐sporulating bacteria. The enzyme behaves as a d‐aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with d‐Ala‐d‐Ala and d‐Ala‐Gly‐Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn‐binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.
Role of thioredoxin reductase in the Yap1p‐dependent response to oxidative stress in Saccharomyces cerevisiae
Authors: Carmel‐Harel Orna , Stearman Robert , Gasch Audrey P. , Botstein David , Brown Patrick O. , Storz Gisela
Published: March 2003

The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2‐dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2–HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole‐genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2‐inducible genes is elevated in Δtrr1 mutants in the absence of external stress. In Δtrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein‐folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Δtrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress‐induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.
Overlapping and distinct roles of the duplicated yeast transcription factors Ace2p and Swi5p
Authors: Doolin Marie‐Therese , Johnson Anthony L. , Johnston Leland H. , Butler Geraldine
Published: March 2003

The yeast transcription factors Ace2p and Swi5p regulate the expression of several target genes involved in mating type switching, exit from mitosis and cell wall function. We describe the analysis of 12 novel targets, some regulated by Ace2p or Swi5p alone and some by both. We show that Ace2p is the major regulator of four genes (CTS1, YHR143W, SCW11 and YER124C). Expression of all four is inhibited by Swi5p. Like Cts1p and Scw11p, the two new Ace2p targets are associated with cell wall metabolism. Yhr143p is localized to the cell wall, and deletion affects cell separation and enhances pseudohyphal growth. Deleting YER124C also affects cell separation and sensitivity to drugs targeted against the cell wall. Expression of PIR1, YPL158C and YNL046W is dependent on Swi5p alone. In contrast, expression of YBR158W, YNL078W and YOR264W is minimized when both ace2 and swi5 are disrupted. We propose that, although Ace2p and Swi5p co‐operate to induce the expression of a subset of genes, some functional divergence has occurred. This results in a delay in the expression of those genes predominantly regulated by Ace2p, compared with those predominantly regulated by Swi5p.
A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC → YojN → RcsB signalling pathway implicated in capsular synthesis and swarming behaviour
Authors: Takeda Shin‐ichiro , Fujisawa Yojiro , Matsubara Masahiro , Aiba Hirofumi , Mizuno Takeshi
Published: March 2003

In this study, we re‐investigated the previously characterized RcsC (sensor His‐kinase) → RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His → Asp phosphorelay, this RcsC → RcsB framework is somewhat puzzling, because RcsC appears to contain both a His‐kinase domain and a receiver domain, but not a histidine (His)‐containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His → Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His‐containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC → YojN → RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.
MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis
Authors: Stahl Claudia , Kubetzko Susanne , Kaps Iris , Seeber Silke , Engelhardt Harald , Niederweis Michael
Published: March 2003

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water‐filled channels in vitro. Immunogold electron microscopy and an enzyme‐linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA‐specific primers and reverse‐transcribed RNA. Detergent extracts of the ΔmspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild‐type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the ΔmspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine‐ and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the ΔmspA mutant. The minimal permeability coefficient of the ΔmspA mutant for glucose was 7.2 × 10−8 cm s−1, which is the lowest value reported so far for bacteria. This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.
Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene
Authors: Gorbatyuk Boris , Marczynski Gregory T.
Published: March 2003

Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6–8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, β‐galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.
The act operon controls the level and time of C‐signal production for Myxococcus xanthus development
Authors: Gronewold Thomas M. A. , Kaiser Dale
Published: March 2003

The C‐signal is a morphogen that controls the assembly of fruiting bodies and the differentiation of myxospores. Production of this signal, which is encoded by the csgA gene, is regulated by the act operon of four genes that are co‐transcribed from the same start site. The act A and act B genes regulate the maximum level of the C‐signal, which never rises above one‐quarter of the maximum wild‐type level of CsgA protein in null mutants of either gene. The act A and act B mutants have the same developmental phenotype: both aggregate, neither sporulates, both prolong rippling. By sequence homology, act A encodes a response regulator, and act B encodes a sigma‐54 activator protein of the NTRC class. The similar phenotypes of act A and act B deletion mutants suggest that the two gene products are part of the same signal transduction pathway. That pathway responds to C‐signal and also regulates the production of CsgA protein, thus creating a positive feedback loop. The act C and act D genes regulate the time pattern of CsgA production, while achieving the same maximum level. An act C null mutant raises CsgA production 15 h earlier than the wild type, whereas an act D null mutant does so 6 h later than wild type. The loop explains how the C‐signal rises continuously from early development to a peak at the time of sporulation, and the act genes govern the time course of that rise.
Conformational changes play a role in regulating the activity of the proline utilization pathway‐specific regulator in Saccharomyces cerevisiae
Authors: Des Etages Shelley Ann G. , Saxena Deepti , Huang Hoching L. , Falvey Darlene A. , Barber Dianna , Brandriss Marjorie C.
Published: March 2003

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA‐bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the ‘on’ conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non‐inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.
Identifying regulators of transcription in an obligate intracellular pathogen: a metal‐dependent repressor in Chlamydia trachomatis
Authors: Wyllie Susan , Raulston Jane E.
Published: March 2003

A prominent feature exhibited by Chlamydia trachomatis growing in an iron‐limiting environment is a differential pattern of protein expression. In many bacteria, iron‐responsive proteins are regulated at the level of transcription by a family of repressors resembling the Escherichia coli ferric uptake regulator (Fur) protein. Although the chlamydial genome sequencing project did not unveil an obvious Fur homologue, a detailed examination indicated five unassigned open reading frames (ORFs) that would encode products with limited sequence homology to Fur. In this report, each chlamydial ORF was engineered in E. coli, and recombinant proteins were examined for functional characteristics resembling Fur. A Fur‐specific polyclonal antiserum revealed that the protein encoded by ORF CT296 shares antigenic cross‐recognition. Moreover, this protein forms dimers in solution in a fashion analogous to E. coli Fur. Further studies confirmed that the product of ORF CT296 is able to (i) complement Fur activity in a mutant strain of E. coli; and (ii) specifically bind to a 19 bp consensus sequence found in promoters of iron‐regulated genes in E. coli. We propose a designation of dcrA (divalent cation‐dependent regulator A) for ORF CT296, which encodes a protein distantly related to E. coli Fur. DcrA represents the first repressor described for this obligate intracellular bacterium.
Exploring the minimal substrate requirements for trans‐cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis
Authors: Hansen Annika , Pfeiffer Thomas , Zuleeg Tilman , Limmer Stefan , Ciesiolka Jerzy , Feltens Ralph , Hartmann Roland K.
Published: March 2003

We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans‐cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5′ flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000‐fold when the 5′ flank was shortened from 3 to 1 nt. Reduction of the 5′ flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt −2 in the single‐stranded 5′ flank to productive interaction is a general feature of A‐ and B‐type bacterial RNase P enzymes. We also showed that an Rp‐phosphorothioate modification at nt −2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single‐stranded RNA oligonucleotides as short as 5 nt, yielding RNase P‐specific 5′‐phosphate and 3′‐OH termini, with measured turnover rates of up to 0.7 min−1. All cleavage sites were at least 2 nt away from the 5′ and 3′ ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.
Role of the nucleoid‐associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli
Authors: Goldberg M. D. , Johnson M. , Hinton J. C. D. , Williams P. H.
Published: March 2003

Virulence gene expression in enteropathogenic Escherichia coli (EPEC) is governed by a combination of environmental factors and virulence regulators. These factors control the expression of the bundle‐forming pili (BFP), intimin, the type III secretion apparatus and the secreted proteins EspA, EspB, EspD and Tir. Expression of the bfp genes occurs for a short period in early exponential phase during growth in tissue culture medium. The nucleoid‐associated regulator protein, Fis, is also expressed transiently during this period. To determine whether Fis was responsible for the growth phase‐dependent expression of bfp, fis was deleted from the EPEC strain E2348/69S. Paradoxically, the Δfis mutant retained the ability to colonize HEp‐2 cells in a characteristic localized adherence pattern, and Fis was found negatively to regulate the expression of BFP. However, the Δfis mutant failed to induce the accretion of filamentous actin, which is associated with attaching and effacing lesions. Using a combination of Western blotting and a novel multiplex primer extension assay (MPEA), we showed that, although the expression of intimin and Tir was not affected, transcription of the LEE4 operon encoding espADB and the virulence activator, Ler, were found to be Fis dependent.