bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeography and conservation genetics of Eld's deer (Cervus eldi)
Authors: Balakrishnan Christopher N. , Monfort Steven L. , Gaur Ajay , Singh Lalji , Sorenson Michael D.
Published: March 2003

Eld's deer (Cervus eldi) is a highly endangered cervid, distributed historically throughout much of South Asia and Indochina. We analysed variation in the mitochondrial DNA (mtDNA) control region for representatives of all three Eld's deer subspecies to gain a better understanding of the genetic population structure and evolutionary history of this species. A phylogeny of mtDNA haplotypes indicates that the critically endangered and ecologically divergentC. eldi eldiis related more closely toC. e. thaminthan toC. e. siamensis, a result that is consistent with biogeographic considerations. The results also suggest a strong degree of phylogeographic structure both between subspecies and among populations within subspecies, suggesting that dispersal of individuals between populations has been very limited historically. Haplotype diversity was relatively high for two of the three subspecies (thaminandsiamensis), indicating that recent population declines have not yet substantially eroded genetic diversity. In contrast, we found no haplotype variation withinC. eldi eldior the Hainan Island population ofC. eldi siamensis, two populations which are known to have suffered severe population bottlenecks. We also compared levels of haplotype and nucleotide diversity in an unmanaged captive population, a managed captive population and a relatively healthy wild population. Diversity indices were higher in the latter two, suggesting the efficacy of well‐designed breeding programmes for maintaining genetic diversity in captivity. Based on significant genetic differentiation among Eld's deer subspecies, we recommend the continued management of this species in three distinct evolutionarily significant units (ESUs). Where possible, it may be advisable to translocate individuals between isolated populations within a subspecies to maintain levels of genetic variation in remaining Eld's deer populations.
Identification of a gene essential for O‐acetylation of the Staphylococcus aureus type 5 capsular polysaccharide
Authors: Bhasin Navneet , Albus Anne , Michon Francis , Livolsi Paul J. , Park Jin‐Sir , Lee Jean C.
Published: March 2003

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (→ 4)‐3‐O‐Ac‐β‐D‐ManNAcAp‐(1 → 4)‐α‐L‐FucNAcp‐(1 → 3)‐β‐D‐FucNAcp‐(1→). Tn918 mutagenesis of strain Reynolds yielded a mutant that produced wild‐type levels of O‐deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL‐LacA‐CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S. aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O‐deacetylated mutant. When cap5H was provided to the mutant in trans, it fully restored CP5 O‐acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.
Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae
Authors: Colmer Jane A. , Fralick Joe A. , Hamood Abdul N.
Published: March 2003

Multidrug‐resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V. cholerae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.
Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae
Authors: Coffey Tracey J. , Enright Mark C. , Daniels Maggie , Morona Judy K. , Morona Renato , Hryniewicz Waleria , Paton James C. , Spratt Brian G.
Published: March 2003

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP‐PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross‐over points appeared to be upstream of dexBwhich flanks one end of the capsular locus, and in six of the variants the other cross‐over point was downstream of aliA, which flanks the other end of the locus. In two strains a recombinational cross‐over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long‐term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.
Isolation and analysis of xlnR, encoding a transcriptional activator co‐ordinating xylanolytic expression in Aspergillus niger
Authors: Van Peij Noël N. M. E. , Visser Jaap , De Graaff Leo H.
Published: March 2003

Complementation by transformation of an Aspergillus niger mutant lacking xylanolytic activity led to the isolation of the xlnR gene. The xlnR gene encodes a polypeptide of 875 amino acids capable of forming a zinc binuclear cluster domain with similarity to the zinc clusters of the GAL4 superfamily of transcription factors. The XlnR‐binding site 5′‐GGCTAAA‐3′ was deduced after electrophoretic mobility shift assays, DNase I footprinting and comparison of various xylanolytic promoters. The importance of the second G within the presumed XlnR binding site 5′‐GGCTAAA‐3′ was confirmed in vitro and in vivo. The 5′‐GGCTAAA‐3′ consensus sequence is found within several xylanolytic promoters of various Aspergillus species and Penicillium chrysogenum. Therefore, this sequence may be an important and conserved cis‐acting element in induction of xylanolytic genes in filamentous fungi. Our results indicate that XlnR is a transcriptional activator of the xylanolytic system in A. niger.
Transcriptional and post‐transcriptional regulation by nickel ofsodNgene encoding nickel‐containing superoxide dismutase fromStreptomyces coelicolorMüller
Authors: Kim Eun‐Ja , Chung Hye‐Jung , Suh Bumsu , Hah Yung Chil , Roe Jung‐Hye
Published: March 2003

A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in severalStreptomycesspp. The gene for NiSOD (sodN ) was cloned fromS.coelicolorMüller using degenerate oligonucleotide probes designed from the N‐terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N‐terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of thesodNgene was determined by S1 mapping and primer extension analysis. Ni2+regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5′ and 3′ ends ofsodNmRNA revealed that Ni2+increased the level of monocistronicsodNmRNA by more than ninefold without changing its half‐life, thus demonstrating that Ni2+regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the clonedsodNgene inS.lividansin the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full‐lengthsodNgene inE.coliproduced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N‐terminal 14 amino acids from thesodNgene allowed the production of active NiSOD inE.coli, indicating that N‐terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator inS.coelicolorcontrollingsodNtranscription and protein processing, as well as acting as a catalytic cofactor.
PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells
Authors: Marenda Marc , Brito Belen , Callard Didier , Genin Stéphane , Barberis Patrick , Boucher Christian , Arlat Matthieu
Published: March 2003

The Ralstonia solanacearum hrp gene cluster is organized in five transcriptional units. Expression of transcriptional units 2, 3 and 4 is induced in minimal medium and depends on the hrp regulatory gene hrpB, which belongs to unit 1. This regulatory gene also controls the expression of genes, such as popAlocated to the left of the hrp cluster. Here, we show that, upon co‐culture with Arabidopsis thaliana and tomato cell suspensions, the expression of the hrp transcriptional units 1, 2, 3 and 4 is induced 10‐ to 20‐fold more than in minimal medium. This induction is not triggered by diffusible signals but requires the presence of plant cells. Moreover, we show that this specific plant cell induction of hrp genes is controlled by a gene, called prhA (plant regulator of hrp genes), located next to popA. This gene codes for a putative protein of 770 amino acids, which shows similarities with TonB‐dependent outer membrane siderophore receptors. Expression of prhA and hrp genes is not regulated by iron status, and we postulate that iron is not the signal sensed by PrhA. In prhA mutants, the induction of hrpB and other hrp genes is abolished in co‐culture with Arabidopsis cells, partially reduced in co‐culture with tomato cells and not modified in minimal medium. prhA mutants are hypoaggressive on Arabidopsis (accessions Col‐0 and Col‐5) but remain fully pathogenic on tomato plants, suggesting that the co‐culture assays mimic the in planta conditions. A model suggesting that PrhA is a receptor for plant specific signals at the top of a novel hrp regulatory pathway is discussed.
The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae
Authors: Didion Thomas , Regenberg Birgitte , Jørgensen Marianne U. , Kielland‐Brandt Morten C. , Andersen Helge A.
Published: March 2003

Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine‐inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D‐leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.
Endonuclease II of coliphage T4: a recombinase disguised as a restriction endonuclease?
Authors: Carlson Karin , Kosturko Linda D.
Published: March 2003

EndoII shares with restriction endonucleases the property of cleaving foreign DNA while leaving the endonuclease‐encoding genome intact, ensuring the survival of one DNA species in the cell. In addition, in vivo EndoII cleaves a specific DNA sequence and cleavage is context dependent. These context effects extend over at least 1000 bp, largely limiting cleavage to once within this distance. Like homing endonucleases, in vivo EndoII recognizes a long, asymmetric and degenerate consensus sequence which has two distinct parts. Recognition of one part of the consensus sequence involves base‐specific bonds, and recognition of the other involves sequence‐dependent helical structure. EndoII fulfils an obvious short‐term survival role in ensuring the dominance of phage DNA in an infected cell, but may also have a long‐term evolutionary role, producing gene‐size fragments of foreign DNA to be enrolled in the phage genetic repertoire.
Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti‐sigma factor FlgM
Authors: Saito Tokuo , Ueno Takamasa , Kubori Tomoko , Yamaguchi Shigeru , Iino Tetsuo , Aizawa Shin‐Ichi
Published: March 2003

Among motile revertants isolated from flagellar hook‐deficient (flgE ) mutants of Salmonella typhimuriumone produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro. This pseudorevertant has an intragenic suppressor, resulting in a two‐amino‐acid substitution (Asp‐Gln→Ala‐Arg) in the C‐terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full‐length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook–basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl‐free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti‐sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.
Serratia marcescens S‐layer protein is secreted extracellularly via an ATP‐binding cassette exporter, the Lip system
Authors: Kawai Eri , Akatsuka Hiroyuki , Idei Akiko , Shibatani Takeji , Omori Kenji
Published: March 2003

The Serratia marcescens Lip exporter belonging to the ATP‐binding cassette (ABC) exporter is known to be involved in signal peptide‐independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several Gram‐negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD. A gene (slaA) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S‐layer) protein. The Lip exporter‐deficient mutants of S. marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S. marcescens. The S‐layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S‐layer secretion, indicating that the S. marcescens S‐layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S‐layer protein via its own secretion system.
Influence of FIS on the transcription from closely spaced and non‐overlapping divergent promoters for an aminoacyl‐tRNA synthetase gene (gltX ) and a tRNA operon (valU ) in Escherichia coli
Authors: Champagne Nathalie , Lapointe Jacques
Published: March 2003

The gltX gene, encoding the glutamyl‐tRNA synthetase (GluRS), and the valU operon, whose transcripts contain three tRNAVal/UAC and one tRNALys/UUU, are adjacent and divergently transcribed. It is the only known case of adjacent genes encoding an aminoacyl‐tRNA synthetase and a tRNA precursor in Escherichia coli. The gltX promoters (P1, P2 and P3) direct the synthesis of transcripts non‐overlapping with and divergent from the one initiated at the valU promoter. We report that their promoter region (250 bp) contains three binding sites for the factor for inversion stimulation (FIS), centred at positions −71, −91 and −112 from the valU transcription initiation site, and that the destruction of any of these sites does not prevent the binding of FIS to the others. As FIS is one of the major positive regulators of stable RNA operons, we have studied its role on gltX and valU transcription. FIS stimulates valU transcription in vitro and about twofold in vivo during steady‐state exponential growth. In contrast, gltX transcription is repressed by the presence of FIS in vitro and about twofold in vivo during growth acceleration when a decrease in GluRS concentration was observed. Under all conditions tested, most of the gltX transcripts start at the P3 promoter. Nested deletions of this regulatory region reveal that the FIS‐dependent repression of the gltX‐P3 promoter is abolished after the removal of the valU promoter, and is not altered by the additional removal of the FIS binding sites; moreover, in vivo transcription from gltX‐P1 and/or gltX‐P2 present on some of these regulatory region variants is modulated by the nature of the upstream region by FIS and is sometimes stronger than that from gltX‐P3. These results show that the strength and the site of gltX transcription initiation are influenced by the upstream region up to and including the valU promoter; furthermore, they indicate that although these adjacent genes are involved in the first step of protein biosynthesis and share cis and trans regulatory elements, their transcription is non‐co‐ordinate.
Ordered expression of ftsQA and ftsZ during the Caulobacter crescentus cell cycle
Authors: Sackett Marcella J. , Kelly Aaron J. , Brun Yves V.
Published: March 2003

The mechanisms by which bacterial cell division and DNA replication are co‐ordinated are still unknown. We have used the easily synchronizable bacterium Caulobacter crescentus to determine when the cell division genes ftsQ and ftsA are transcribed during the DNA replication cycle and to compare their transcription with that of ftsZ. Unlike the situation in Escherichia coli, transcription of ftsQ and ftsA does not extend into ftsZ in Caulobacter. ftsQ and ftsA are co‐transcribed by a strong promoter, PQA, present within the end of the ddl gene upstream of ftsQ. Transcription of PQA is turned on at the end of the DNA replication period, coincident with the end of the ftsZ transcription period. ftsA is also transcribed by another promoter, PA, present between ftsQ and ftsA. PA transcription is ≈10 times weaker than PQA and occurs during the DNA replication period. Transcription of ftsA by PA is sufficient for cell viability, but is not sufficient for normal cell division. When the transcription of ftsA is increased constitutively, cell division is inhibited and stalks are synthesized at aberrant positions. Thus, transcription of ftsA and ftsZ mimics their order of action in Caulobacter and proper transcription of ftsA has to be maintained for normal cell division and differentiation.
A rolling circle replication initiator protein with a nucleotidyl‐transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi
Authors: Marsin Stéphanie , Forterre Patrick
Published: March 2003

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking‐closing activity in vitro on single‐stranded oligonucleotides containing the putative double‐stranded replication origin sequence of pGT5. Gel shift analyses on single‐stranded oligonucleotides indicate that Rep75 recognizes the single‐stranded DNA region upstream of the nicking site via non‐covalent interaction and remains covalently linked to the 5′‐phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3′‐OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.
EspB and EspD require a specific chaperone for proper secretion from enteropathogenicEscherichia coli
Authors: Wainwright Leslie A. , Kaper James B.
Published: March 2003

EnteropathogenicEscherichia coliuses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation incesDabolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.
Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli
Authors: Wolff Carmel , Nisan Israel , Hanski Emanuel , Frankel Gad , Rosenshine Ilan
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children. EPEC induces the formation of actin pedestal in infected epithelial cells. A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals. We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB. Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB–CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle‐forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB–CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB–CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.
Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence
Authors: Engbring J. A. , Alderete J. F.
Published: March 2003

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single‐copy AP33 genes and suggested a semi‐conservative genomic arrangement between T. vaginalis isolates. Analysis of full‐length sequences determined that each contained a 930 bp open reading frame encoding a protein of approximately 33 000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N‐terminal protein sequencing established the presence of leader peptides. Each of the three full‐length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl‐CoA synthetase α‐subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host–parasite relationship.
High‐frequency intracellular infection and erythrogenic toxin A expression undergo phase variation in M1 group A streptococci
Authors: Cleary P. Patrick , McLandsborough Lynne , Ikeda Leo , Cue David , Krawczak Jim , Lam Hong
Published: March 2003

A clonal variant of serotype M1 group A streptococcus, strain 90‐131, disseminated to several continents, where it was associated with severe systemic infections and toxic shock. Although this strain harbours the speA gene and is efficiently internalized by human epithelial cells, clinical isolates often fail to express the erythrogenic toxin under laboratory growth conditions. Cultures of strain 90‐131 were observed to phase vary between small, dry, compact and larger, more mucoid colonies. The former were shown to be poorly internalized by epithelial cells. Analysis of RNA by Northern hybridization demonstrated that the emm1, hasA and speA genes were weakly transcribed in cultures derived from the small colonies and highly transcribed in those derived from the large colonies. An insertion mutation in mga (the multigene activator) downregulated the invasion of epithelial cells and the transcription of emm1 and hasA, but had little impact on the transcription of speA. These are the first data to suggest the existence of a common regulatory circuit linking intracellular invasion, M protein, hyaluronic acid capsule and erythrogenic toxin expression by group A streptococcus. Moreover, the genetic instability of toxin expression exhibited by this serotype may impact on laboratory studies that attempt to associate toxin production with toxic shock.
The redox‐ and fixed nitrogen‐responsive regulatory protein NIFL from Azotobacter vinelandii comprises discrete flavin and nucleotide‐binding domains
Authors: Söderbäck Erik , Reyes‐Ramirez Franscisca , Eydmann Trevor , Austin Sara , Hill Susan , Dixon Ray
Published: March 2003

Azotobacter vinelandii NIFL is a nitrogen fixation‐specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N‐terminal domain and a C‐terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide‐binding sites in the C‐terminus, whereas ADP protects the entire C‐terminal domain. NIFL has an apparent Kd of 130 μM for ATP and 16 μM for ADP. The purified N‐terminal domain has an identical UV/visible absorption spectrum to the wild‐type protein and is reduced by sodium dithionite, demonstrating that it is a flavin‐binding domain. The isolated N‐terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C‐terminal region, including the nucleotide‐binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S‐motif (PAS‐like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen‐sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.
Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis
Authors: Parsons Linda M. , Jankowski Craig S. , Derbyshire Keith M.
Published: March 2003

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc2155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.
Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation
Authors: Moe Paul C. , Blount Paul , Kung Ching
Published: March 2003

mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E. coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild‐type E. coli channel, and a C‐terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.
Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator
Authors: Ingmer Hanne , Miller Christine A. , Cohen Stanley N.
Published: March 2003

We identified a gene (dpiAdestabilizer of plasmid inheritance) which, when overexpressed in Escherichia coli, destabilizes the inheritance of pSC101 and other iteron‐containing plasmids as disparate as mini‐F and RK6 but not the inheritance of P1, RSF1010 and ColD. These effects of DpiA, which functions like an effector protein for a previously undescribed two‐component signal transduction system, were reduced by mutations known to promote pSC101 replication and partitioning. dpiB, a gene encoding the putative histidine kinase of this two‐component system, is located immediately 5′ to dpiA and adjacent to a DpiA‐induced target promoter that transcribes genes having homology to citrate lyase operon genes, citC, citD and citE, of Klebsiella pneumoniae. Disruption of dpiB reversed or reduced the effect of DpiA overproduction on pSC101 inheritance. A second DpiA target, the promoter for a gene (appY ) implicated in E. coli's response to anaerobiosis, is repressed by DpiA. A mutation in dpiA at a site commonly conserved and phosphorylated in two‐component system effector proteins abolished the effects of DpiA overproduction on pSC101 inheritance and negative regulation of appY expression. Our findings suggest a possible mechanism by which environmental and/or cellular stimuli may influence plasmid inheritance.
Multiple cis‐acting sites positively regulate Escherichia coli nrd expression
Authors: Jacobson Blake A. , Fuchs James A.
Published: March 2003

Regulation of nrd expression in Escherichia coli by cis‐acting elements was found to be more complex than previously reported. At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT‐rich region, an inverted repeat and a 10 bp site between the AT‐rich region and the inverted repeat. Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression. As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis‐inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states. As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle‐regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites. Site‐directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT‐rich region results from deletion of both the Fis binding site and the AT‐rich region.
Structural homologues PII and Pz of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen‐dependent functions
Authors: De Zamaroczy Miklos
Published: March 2003

PII (glnB ) is a signal transduction protein that in Azospirillum brasilense is specifically required for nitrogen fixation. Little is known about whether and how its homologue Pz (glnZ ) participates in the regulation of cellular functions. In this study, we have shown the regulatory action of the two proteins by analysing the relevant single and double null‐mutant strains. The transcription of glnZ is monocistronic, and it starts mainly from a σ54‐dependent promoter, activated by NtrC. glnZ expression is dependent on the ntr system, even under conditions of nitrogen excess, and is greatly enhanced in the presence of aspartate. Pz is uridylylated in response to nitrogen limitation, like PII, although different amounts of the two proteins are synthesized. PII is required for the dephosphorylation of NtrC. Thus, in the absence of PII, the repression of nitrate assimilation is not promoted, which, in turn, leads to a high rate of ammonium excretion. Unexpectedly, PII and Pz proteins are not essential for the reversible modification of glutamine synthetase. (Methyl)ammonium transport into the cell is negatively regulated by Pz. The growth of a double‐mutant strain (glnB ::kan; glnZ ::Ω ) is drastically disabled, although wild‐type growth is restored by complementation with either glnB or glnZ. We conclude that PII and Pz, despite their structural similarity, are involved in different regulatory processes, except for that required for cell growth.
Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11
Authors: Roberts Julia E. , Watters James W. , Ballard Jimmy D. , Dietrich William F.
Published: March 2003

The lethal factor (LF) toxin that is produced by Bacillus anthracis plays an important role in the pathogenesis of anthrax. LF has mononuclear phagocyte‐specific intoxicating effects that are not well understood. We have identified genetic differences in inbred mouse strains that determine whether their cultured macrophages are susceptible to the cytolytic effect of LF intoxication. Our identification of resistant and susceptible mouse strains enabled us to analyse crosses between these strains and to map a single responsible gene (called Ltx1 ) to chromosome 11. Ltx1 probably influences intoxication events that occur after the delivery of LF to the cytosol, as all mouse macrophages are killed by polypeptides containing the catalytic domain of Diphtheria toxin fused to the domain of LF required for cytosolic transport. Furthermore, the susceptibility phenotype is dominant to resistance, suggesting that resistance is caused by an absence of or polymorphism in a molecule that acts jointly with, or downstream of, the activity of LF. Our mapping of Ltx1 is a crucial first step in its positional cloning, which will provide more information about the mechanism of LF intoxication.
A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli
Authors: Gardina Paul J. , Bormans Arjan F. , Manson Michael D.
Published: March 2003

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate‐loaded maltose‐binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand‐binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid‐borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (Δtar ). However, when Tar proteins with complementing sets of mutations were co‐expressed from compatible plasmids, the resulting heterodimeric receptors enabled Δtar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the ‘same’ orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the ‘opposite’ orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the ‘opposite’ binding orientation. When aspartate and MBP bind in the ‘same’ orientation, they compete for signalling through one subunit. In the wild‐type Tar dimer, aspartate and MBP can bind in either the ‘same’ or the ‘opposite’ orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar+ cells.
Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo
Authors: Regnacq Matthieu , Hewitt Eric , Allen Jeannette , Rosamond John , Stirling Colin J.
Published: March 2003

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98–171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N‐terminal and C‐terminal domains (residues 1–97 and 172–273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C‐terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65‐1 mutation in S. cerevisiae. In order to examine the role of the N‐ and C‐ terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro. These studies indicate that a minimal Sec65p comprising residues 76–209, which includes the entire central SRP19‐like domain, is sufficient for SRP function in yeast.
Non‐specific, general and multiple stress resistance of growth‐restricted Bacillus subtilis cells by the expression of the σB regulon
Authors: Hecker Michael , Völker Uwe
Published: March 2003

Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the σB‐dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of σB and knowledge of a very large number of general stress genes controlled by σB, first insights into the physiological role of this non‐specific stress response have been obtained only very recently. To explore the physiological role of this regulon, we and others identified σB‐dependent general stress genes and compared the stress tolerance of wild‐type cells with mutants lacking σB or general stress proteins. The proteins encoded by σB‐dependent general stress genes can be divided into at least five functional groups that most probably provide growth‐restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non‐specific resistance to oxidative stress, which requires the σB‐dependent dps gene encoding a DNA‐protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of σB‐mediated general stress resistance in growth‐arrested aerobic Gram‐positive bacteria. Other general stress genes have both a σB‐dependent induction pathway and a second σB‐independent mechanism of stress induction, thereby partially compensating for a σB deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as clpP or clpC, cause extreme sensitivity to salt or heat.
The Escherichia coli threonyl‐tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation
Authors: Sacerdot Christine , Caillet Joel , Graffe Monique , Eyermann Flore , Ehresmann Bernard , Ehresmann Chantal , Springer Mathias , Romby Pascale
Published: March 2003

The expression of the gene encoding Escherichia coli threonyl‐tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single‐stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non‐contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single‐stranded sequence in domain 3. These two regions are brought into close proximity by a 38‐nucleotide‐long hairpin structure (domain 2). This domain, although adjacent to the 5′ edge of the SD sequence, does not inhibit ribosome binding as long as the single‐stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.
Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase
Authors: Kang Choong Min , Vijay Kamni , Price Chester W.
Published: March 2003

The RsbT serine kinase has two known functions in the signal transduction pathway that activates the general stress factor σB of Bacillus subtilis. First, RsbT can phosphorylate and inactivate its specific antagonist protein, RsbS. Second, upon phosphorylation of RsbS, RsbT is released to stimulate RsbU, a PP2C phosphatase, thereby initiating a signalling cascade that ultimately activates σB. Here we describe a mutation that separates these two functions of RsbT. Although the mutant RsbT protein had essentially no kinase activity, it still retained the capacity to stimulate the RsbU phosphatase in vitro and to activate σB when overexpressed in vivo. These results support the hypothesis that phosphatase activation is accomplished via a long‐lived interaction between RsbT and RsbU. In contrast, RsbT kinase activity was found to be integral for the transmission of external stimuli to σB. Thus, one route by which environmental stress signals could enter the σB network is by modulation of the RsbT kinase activity, thereby controlling the magnitude of the partner switch between the RsbS–RsbT complex and the RsbT–RsbU complex.