bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the A2–A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection
Authors: Zhang Wen‐Wei , Matlashewski Greg
Published: March 2003

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2–A2rel gene clusters, the 5′ and 3′ DNA sequences flanking the A2–A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2–A2rel gene clusters knock‐out mutants, it was not possible to delete all the A2–A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2‐expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.
RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II
Authors: Cairrão Fátima , Chora Ângelo , Zilhão Rita , Carpousis Agamemnon J. , Arraiano Cecília M.
Published: March 2003

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.
A guided tour: small RNA function in Archaea
Authors: Dennis Patrick P. , Omer Arina , Lowe Todd
Published: March 2003

In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2′‐O‐ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis. Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature. The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs. The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature. We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2′‐O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.
The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues
Authors: Skouloubris Stéphane , Labigne Agnès , De Reuse Hilde
Published: March 2003

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short‐chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two‐step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys‐165 in AmiE and Cys‐166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.
Sut1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p–Tup1p repression in Saccharomyces cerevisiae
Authors: Régnacq Matthieu , Alimardani Parissa , Moudni Brahim El , Bergès Thierry
Published: March 2003

SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn[II]2Cys‐6 family. It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells. A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction. Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters. Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene. We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)–Tup1p co‐repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p. Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p. In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.
Spatial and temporal sequence of capsule construction in Cryptococcus neoformans
Authors: Pierini Lynda M. , Doering Tamara L.
Published: March 2003

The pathogenic yeast Cryptococcus neoformans is distinguished by an extensive polysaccharide capsule, which impedes host defences and is absolutely required for fungal virulence. Despite the biological importance of the capsule, nothing is known about how it is assembled. Substantial capsule growth occurs in two distinct situations relevant to cryptococcal pathogenesis: formation of new buds and induction of capsule on mature cells. We developed pulse–chase protocols to examine these events in a dynamic way using a variety of microscopy techniques. We show that the capsule overlying buds is newly synthesized and differs physically from the corresponding parental material. New capsule formed by mature cells upon induction of synthesis is added at the inner aspect of the existing structure, displacing pre‐existing material outwards. Surprisingly, new polysaccharide material is also deposited throughout the capsule, yielding a progressively denser structure. These results yield the first model of capsule synthesis and open new lines of investigation into the underlying mechanisms.
Horizontal gene transfer and host specificity of beta‐haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb
Authors: Franken C. , Haase G. , Brandt C. , Weber‐Heynemann J. , Martin S. , Lämmler C. , Podbielski A. , Lütticken R. , Spellerberg B.
Published: March 2003

Beta‐haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.
Identification of TogMNAB, an ABC transporter which mediates the uptake of pectic oligomers in Erwinia chrysanthemi 3937
Authors: Hugouvieux‐Cotte‐Pattat Nicole , Blot Nicolas , Reverchon Sylvie
Published: March 2003

The bacterium Erwinia chrysanthemi, which causes soft rot disease on various plants, is able to use pectin as a carbon source for growth. Knowledge of the critical step in pectin catabolism which allows the entry of pectic oligomers into the cells is scarce. We report here the first example of a transport system involved in the uptake of pectic oligomers. The TogMNAB transporter of E. chrysanthemi is a member of the ATP‐binding cassette (ABC) superfamily. TogM and TogN are homologous to the inner membrane components, TogA exhibits the signature of ABC ATPases and TogB shows similarity with periplasmic ligand‐binding proteins. The TogMNAB transporter is a new member of the carbohydrate uptake transporter‐1 family (CUT1, TC no. 3.1.1), which is specialized in the transport of complex sugars. The four genes, togM, togN, togA and togB, are apparently co‐transcribed in a large operon which also includes the pectate lyase gene pelW. The transcription of the tog operon is induced in the presence of pectic derivatives and is affected by catabolite repression. It is controlled by the KdgR repressor and the CRP activator. The TogMNAB system is able to provide Escherichia coli with the ability to transport oligogalacturonides. In E. chrysanthemi, the TogMNAB system seems to play a major role in switching on the induction of pectin catabolism. TogB also acts as a specific receptor for chemotaxis towards oligogalacturonides. The decreased capacity of maceration of a togM mutant indicates the importance of transport and/or attraction of oligogalacturonides for E. chrysanthemi pathogenicity.
Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm
Authors: Journet Laure , Bouveret Emmanuelle , Rigal Alain , Lloubes Roland , Lazdunski Claude , Bénédetti Hélène
Published: March 2003

Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N‐terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N‐terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N‐terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic‐exposed Tol proteins are therefore a general principle required for group A colicin translocation.
Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant
Authors: Yang Wenli , Tabancay Angel P. , Urano Jun , Tamanoi Fuyuhiko
Published: March 2003

Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1–, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G‐protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb‐CVIL) that can bypass farnesylation. In contrast, a farnesylation‐defective mutant of SpRheb (SpRheb‐SVIA) is incapable of restoring the normal cell cycle profile to the cpp1– mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb– disruption can be complemented by the introduction of wild‐type sprheb+. The complementation is dependent on farnesylation, as the farnesylation‐defective SpRheb‐SVIA mutant is incapable of complementing the sprheb– disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb– disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. Our findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process.
RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon
Authors: Ramírez‐Romero Miguel A. , Téllez‐Sosa Juan , Barrios Humberto , Pérez‐Oseguera Angeles , Rosas Vania , Cevallos Miguel A.
Published: March 2003

The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans‐incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site‐directed mutagenesis of the putative −35 and −10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.
SigB, an RNA polymerase sigma factor required for osmoprotection and proper differentiation of Streptomyces coelicolor
Authors: Cho You‐Hee , Lee Eun‐Jin , Ahn Bo‐Eun , Roe Jung‐Hye
Published: March 2003

A gene (sigB) encoding an alternative sigma factor σB in Streptomyces coelicolor A3(2) was isolated and characterized. It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilisσB. The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order. RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon). Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of σB protein. An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions. Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts. Both catBp and sigBp1 promoters were recognized specifically by σB‐containing RNA polymerase in vitro. Disruption of the sigB gene abolished not only the differentiation‐associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of σB. The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production. Therefore, we conclude that σB ensures the proper differentiation and osmoprotection of S. coelicolor cells, primarily via regulation of the expression of catalase B.
Orientational control of fimE expression in Escherichia coli
Authors: Sohanpal Baljinder K. , Kulasekara Hemantha Don , Bonnen Alice , Blomfield Ian C.
Published: March 2003

Phase‐variable expression of type 1 fimbriae is, in part, controlled by site‐specific DNA inversion of the fim switch in Escherichia coli. Of the two fim recombinases (FimB and FimE) that catalyse the inversion reaction, FimE exhibits a strong bias for phase switching from the ON to the OFF orientation. The specificity associated with fimE is the result of two different mechanisms: (i) FimE exhibits a preference for the invertible element in the ON orientation as substrate for recombination; (ii) the invertible element in the OFF orientation acts in cis to inhibit recombinase activity (orientational control). We show here that the invertible element negatively regulates fimE, even though expression of a fimE–lacZYA transcriptional fusion is unaffected by orientational control. The fimE transcript extends into the invertible region and hence switch ON‐specific and switch OFF‐specific mRNA contain different sequences. Furthermore, we show that orientational control is suppressed by the insertion of a structured RNA (tRNAGly) between fimE and the fim switch, indicating that the switch OFF‐specific mRNA is inactivated by 3′ to 5′ degradation. Analysis of the fim switch reveals that it contains two inhibitory elements that exert orientational control independently.
Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the σR regulon
Authors: Paget Mark S. B. , Molle Virginie , Cohen Gerald , Aharonowitz Yair , Buttner Mark J.
Published: March 2003

In the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2), the thiol‐disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, σR, and its cognate anti‐sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for σR, thereby releasing σR to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for σR target promoters to identify 27 new σR target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin‐like protein and enzymes involved in the biosynthesis of the low‐molecular‐weight thiol‐containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the σR targets. Three σR target genes encode ribosome‐associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another σR target gene was found to be a novel RNA polymerase‐associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the σR homologue in the actinomycete pathogen Mycobacterium tuberculosis.
Translation at higher than an optimal level interferes with coupling at an intercistronic junction
Authors: Yu Jae‐Sung , Madison‐Antenucci Susan , Steege Deborah A.
Published: March 2003

In pairs of adjacent genes co‐transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single‐stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild‐type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine–Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.
Mutational analysis of F‐pilin reveals domains for pilus assembly, phage infection and DNA transfer
Authors: Manchak J. , Anthony . G. , Frost Laura S.
Published: March 2003

The F‐pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F‐pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F‐pilus tip, and the RNA phages, R17 and Qβ,
which attach to different sites exposed on the sides of the pilus. R17 has been shown
to undergo eclipse, or capsid release, outside the cell on pili attached to cells.
New and existing mutants of traA combined with natural variants of F‐pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F‐pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45–60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.
Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C‐terminal two amino acids of the A2 peptide in the context of the appropriate B pentamer
Authors: Melton‐Celsa Angela R. , Kokai‐Kun John F. , O’Brien Alison D.
Published: March 2003

Shiga toxins (Stx) are potent ribosome‐inactivating toxins that are produced by Shigella dysenteriae type 1 or certain strains of Escherichia coli. These toxins are composed of one A subunit that can be nicked and reduced to an enzymatically active A1(≈27 kDa) and an A2 peptide (≈4 kDa) as well as a pentamer of B subunits (≈7 kDa/monomer) that binds the eukaryotic cell. Purified Shiga toxin type 2d is activated 10‐ to 1000‐fold for Vero cell toxicity by preincubation with mouse or human intestinal mucus or purified mouse elastase, whereas Stx2, Stx2c, Stx2e and Stx1 are not activatable. E. coli strains that produce the activatable Stx2d are more virulent in a streptomycin (str)‐treated mouse model of infection [lethal dose 50% (LD50) = 101] than are E. coli strains that produce any other type of Stx (LD50= 1010). To identify the element(s) of Stx2d that are required for mucus‐mediated activation, toxin genes were constructed such that the expressed mutant toxins consisted of hybrids of Stx2d and Stx1, Stx2 or Stx2e, contained deletions of up to six amino acids from the C‐terminus of the A2 of Stx2d or were altered in one or both of the two amino acids of the A2 of Stx2d that represent the only amino acid differences between the activatable Stx2d and the non‐activatable Stx2c. Analysis of these mutant toxins revealed that the A2 portion of Stx2d is required for toxin activation and that activation is abrogated if the Stx1 or Stx2e B subunit is substituted for the Stx2d B polypeptide. Furthermore, mass spectrometry performed on buffer‐ or elastase‐treated Stx2d indicated that the A2 peptide of the activated Stx2d was two amino acids smaller than the A2 peptide from buffer‐treated Stx2d. This finding, together with the toxin hybrid results, suggests that activation involves B pentamer‐dependent cleavage by elastase of the C‐terminal two amino acids from the Stx2d A2 peptide.
Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli
Authors: Martin Robert G. , Gillette William K. , Martin Nicholas I. , Rosner Judah L.
Published: March 2003

Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator–DNA–RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator–DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA–DNA–RNP and SoxS–DNA–RNP complexes are more stable than the corresponding Rob–DNA–RNP complex, although the binary Rob–DNA complex is often more stable than the corresponding MarA– or SoxS–DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/ Rob system. We suggest that activator–RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.
Novel gene products associated with NdhD3/D4‐containing NDH‐1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942
Authors: Maeda Shin‐ichi , Badger Murray R. , Price G. Dean
Published: March 2003

Cyanobacteria possess light‐dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3– and may involve a protein‐mediated carbonic anhydrase (CA)‐like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low‐CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light‐dependent CO2 hydration and CO2 uptake reactions; co‐inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH‐1) complexes. The ChpX system has a low affinity for CO2 and is de‐pendent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3–, that is coupled electron flow within the NDH‐1 complex on the thylakoid membrane.
Co‐ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG
Authors: Noubir Sanaâ , Luque Ignacio , Alda Jesús A. G. Ochoa de , Perewoska Irène , Marsac Nicole Tandeau de , Cobley John G. , Houmard Jean
Published: March 2003

In the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red‐light‐grown cells as an alkaline phosphatase‐sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli‐overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co‐transcription of both genes was demonstrated by reverse transcription (RT)‐PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red‐light shifts. RcaD seems to be a co‐activator co‐ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.
Salmonella enterica serotype Typhimurium ShdA is an outer membrane fibronectin‐binding protein that is expressed in the intestine
Authors: Kingsley Robert A. , Santos Renato L. , Keestra A. Marijke , Adams L. Garry , Bäumler Andreas J.
Published: March 2003

The shdA gene is the only determinant known to be required for persistence of Salmonella enterica serotype Typhimurium (S. Typhimurium) in the murine caecum and for efficient and prolonged shedding of the organism with the faeces. To study the biological activity of the ShdA protein, we examined its expression and binding activity. ShdA was not detected with anti‐ShdA antiserum in S. Typhimurium strain ATCC14028 grown in vitro, suggesting that this protein is not expressed under standard conditions of bacterial cultivation in the laboratory. However, in mice infected with S. Typhimurium, an immunofluorescence signal detected with anti‐ShdA antiserum co‐localized with that generated by anti‐O4 antiserum in thin sections from the caecum. Expression of the cloned shdA gene from the T7 promoter in vitro resulted in detection of ShdA in the outer membrane of S. Typhimurium and in binding of fibronectin to the bacterial surface. Binding of purified glutathione‐S‐transferase (GST)–ShdA fusion protein to fibronectin was dose dependent and could be partially inhibited by preincubation with antifibronectin antibodies. GST–ShdA bound to connective tissue and the basement membrane in thin sections from the murine caecum in situ. A similar labelling pattern was produced when thin sections of the murine caecum were stained with antifibronectin antiserum. Collectively, these data demonstrate that ShdA is a surface‐localized, fibronectin‐binding protein whose expression is induced in vivo in the murine caecum, a tissue in which a cognate receptor of this outer membrane protein is expressed.
Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication
Authors: Masson Ivan Le , Saveanu Cosmin , Chevalier Anne , Namane Abdelkader , Gobin Renée , Fromont‐Racine Micheline , Jacquier Alain , Mann Carl
Published: March 2003

Mps2 (monopolar spindle protein) is a coiled‐coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two‐hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled‐coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2–Spc24 interaction by co‐immunoprecipitation in vivo
and by the in vitro interaction of recombinant proteins. Previous two‐hybrid
screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting
partners, and we verified these interactions in vivo by purification of TAP‐tagged
derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com‐ponents required for chromosome segregation. The Mps2–Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.
Transferrin binding in Staphylococcus aureus: involvement of a cell wall‐anchored protein
Authors: Taylor John M. , Heinrichs David E.
Published: March 2003

The ability to gain access to iron is pivotal for bacterial pathogens during infection. Although much is known about iron acquisition systems in Gram‐negative bacteria, comparatively little is known about how Gram‐positive pathogens access iron from host iron sources. A previous study showed that, in the Gram‐positive human pathogen Staphylococcus aureus, a cell surface‐associated glyceraldehyde‐3‐phosphate

dehydrogenase (GAPDH) enzyme (Gap, or Tpn) is capable of binding human transferrin,

representing a potential means by which this bacterium is able to access iron in

vivo. We have investigated this property of S. aureus further and shown

that, in S. aureus RN6390, GAPDH is expressed on the S. aureus cell

surface independent of exogenous iron concentrations, and that overexpressed and

purified Gap, although retaining GAPDH activity, has no affinity for human transferrin.

Moreover, although a S. aureus gap mutant was devoid of surface‐associated

and cytoplasmic GAPDH activity, it retained the ability to bind human transferrin,

equivalent to wild type. We concluded from these results that the Gap protein is

not involved in S. aureus binding to human transferrin. We identified the

transferrin‐binding protein as a novel cell wall‐anchored protein, designated StbA

for staphylococcal transferrin‐binding protein A, which shared

no significant similarities with any other bacterial transferrin‐binding proteins.

StbA contained a C‐terminal cell wall‐anchoring motif (LPKTG), and expression of

StbA in the cell wall was strictly controlled by exogenous iron concentrations. The

stbA gene is found within a 7 kb region in the S. aureus chromosome

that contains a total of six iron‐regulated genes. Immediately downstream from stbA

is an iron‐regulated gene whose product was predicted to be another cell wall‐anchored

protein with no significant similarity to proteins with characterized functions.

Transcribed in the opposite direction from stbA is a four‐gene operon whose expression is also regulated by iron. While the deduced products of the first two genes lack similarity to known proteins, the last two genes encode, respectively, putative lipoprotein and permease components of an ABC transporter that shares significant similarities with several iron(III) ABC transporters in a variety of bacteria.
Developmental switch of S‐layer protein synthesis in Bacillus anthracis
Authors: Mignot Tâm , Mesnage Stéphane , Couture‐Tosi Evelyne , Mock Michèle , Fouet Agnès
Published: March 2003

Adjustment of the synthesis of abundant protein to the requirements of the cell involves processes critical to the minimization of energy expenditure. The regulation of S‐layer genes might be a good model for such processes because expression must be controlled, such that the encoded proteins exactly cover the surface of the bacterium. Bacillus anthracis has two S‐layer genes, sap and eag, encoding

the S‐layer proteins Sap and EA1 respectively. We report that the production and

surface localization of Sap and EA1 are under developmental control, suggesting that

an exponential phase ‘Sap layer’ is subsequently replaced by a stationary

phase ‘EA1 layer’. This switch is controlled at the transcriptional

level: sap is most certainly transcribed by RNA polymerase containing σA,

whereas eag expression depends on σH. More importantly,

Sap is required for the temporal control of eag, and EA1 is involved in strict

feedback regulation of eag. This control may be direct because both S‐layer

proteins bind, in vitro, the eag promoter, specifically suggesting that they might act as transcriptional repressors.
The repressor for an organic peroxide‐inducible operon is uniquely regulated at multiple levels
Authors: Mongkolsuk Skorn , Panmanee Warunya , Atichartpongkul Sopapan , Vattanaviboon Paiboon , Whangsuk Wirongrong , Fuangthong Mayuree , Eiamphungporn Warawan , Sukchawalit Rojana , Utamapongchai Supa
Published: March 2003

ohrR encodes a novel organic peroxide‐inducible transcription repressor,
and we have demonstrated that ohrR is regulated at the transcriptional and
the post‐transcriptional levels. Primer extension results show that ohrR transcription
initiates at the A residue of the ATG translation initiation codon for the ohrR
coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1)
has high homology to the consensus sequence for Xanthomonas promoters, which
is reflected in the high in vivo promoter activity of P1. Deletion of a 139
bp fragment containing the P1 promoter showed that the sequences upstream of –35
regions were required for neither the promoter activity nor OhrR autoregulation.
In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting
of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands.
The protected regions include the –35 and –10 regions of P1. We suggest
that OhrR represses gene expression by blocking RNA polymerase binding to the promoter.
There are two steps in the post‐transcriptional regulation of ohrR, namely
differential stability and inefficient translation of the mRNA. The bicistronic ohrR–ohr
mRNA was highly labile and underwent rapid processing in vivo to give only
stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore,
the ohrR mRNA was inefficiently translated. We propose that, in uninduced
cells, the concentration of OhrR is maintained at low levels by the autoregulation
mechanism at the transcriptional levels and by the ohrR mRNA instability coupled
with inefficient translation at the post‐transcriptional level. Upon exposure to
an organic peroxide, the compound probably interacts with OhrR and prevents it from
repressing the P1 promoter, thus allowing high‐level expression of the ohrR–ohr
operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per‐oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.
Structural mechanisms of multidrug recognition and regulation by bacterial multidrug transcription factors
Authors: Schumacher Maria A. , Brennan Richard G.
Published: March 2003

The increase in bacterial resistance to multiple drugs represents a serious and growing health risk. One component of multidrug resistance (MDR) is a group of multidrug transporters that are often regulated at the transcriptional level by repressors and/or activators. Some of these transcription factors are also multidrug‐binding proteins, frequently recognizing the same array of drugs that are effluxed by the transporters that they regulate. How a single protein can recognize such chemically disparate compounds is an intriguing question from a structural standpoint and an important question in future drug development endeavours. Unlike the multidrug transporters, the cytosolic multidrug‐binding regulatory proteins are more tractable systems for structural analyses. Here, we describe recent crystallographic studies on MarR, BmrR and QacR, three bacterial transcription regulators that are also multidrug‐binding proteins. Although our understanding of multidrug binding and transcriptional regulation by MarR is in its initial stages, the structure of a BmrR–TPP+–DNA complex has revealed important insights into the novel transcription activation mechanism of the MerR family, and the structures of a QacR–DNA complex and QacR bound to six different drugs have revealed not only the mechanism of induction of this repressor but has afforded the first view of any MDR protein bound to multiple drugs.
Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non‐cellulosomal endoglucanase EngD
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H.
Published: March 2003

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non‐cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a com‐bination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.
Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formation
Authors: Fox H. , Hickey P. C. , Fernández‐Ábalos J. M. , Lunness P. , Read N. D. , Doonan J. H.
Published: March 2003

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimGPP1 in mitosis and hyphal growth and are highly dynamic. BIMG‐GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG‐GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG‐GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.
Agrobacterium type IV secretion is a two‐step process in which export substrates associate with the virulence protein VirJ in the periplasm
Authors: Pantoja Mario , Chen Lishan , Chen Yuching , Nester Eugene W.
Published: March 2003

Type IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems. Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear. In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ. Additionally, these proteins co‐precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein. Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two‐step process for type IV secretion in Agrobacterium. Our analyses of protein–protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.
Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl‐lipid desaturase
Authors: Cybulski Larisa E. , Albanesi Daniela , Mansilla María C. , Altabe Silvia , Aguilar Pablo S. , De Mendoza Diego
Published: March 2003

Summary The Des pathway of Bacillus subtilis regulates the expression of the acyl‐lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids (UFAs) from saturated phospholipid precursors. Previously, we showed that the master switch for the Des pathway is a two‐component regulatory system composed of a membrane‐associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. Activation of this pathway takes place when cells are shifted to low growth temperature. Here, we report on the mechanism by which isoleucine regulates the Des pathway. We found that exogenous isoleucine sources, as well as its α‐keto acid derivative, which is a branched‐chain fatty acid precursor, negatively regulate the expression of the des gene at 37°C. The DesK–DesR two‐component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37°C. Fatty acid profiles strongly indicate that isoleucine affects the signalling state of the DesK sensor protein by dramatically increasing the incorporation of the lower‐melting‐point anteiso‐branched‐chain fatty acids into membrane phospholipids. We propose that both a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism. Thus, the Des pathway would provide a novel mechanism to optimize membrane lipid fluidity at a constant temperature.