bluuebunny/crossref_metadata_2025_split huggingface dataset:
Salmonella as an intracellular parasite
Authors: Finiay B. B. , Falkow S. 
Published: October 2006

SummarySalmonella species are facultative intracellular parasites, capable of penetrating (invading), surviving, and often multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. These processes are essential for virulence, and involve both bacterial and host cell products. The use of cultured eukaryotic cells and other model systems has facilitated the study of bacterial‐host cell interactions, and has led to a better understanding of the genetic and molecular basis of Salmonella pathogenicity.
The expression of the DNA ligase gene of Escherichia coli is stimulated by relaxation of chromosomal supercoiling
Authors: Liebart J. C. , Paolozzi L. , Camera M. G. , Pedrini A. M. , Ghelardini P. 
Published: October 2006

SummaryMany genes of Escherichia coli have been shown to be sensitive to DNA superhelicity. The superhelicity of the chromosome is itself also supercoiling‐dependent. We have developed a general strategy for investigating how a particular gene responds to changes in DNA topology. This approach is used to study the E. coli ligase gene. The thermosensitivity of the E. coli ligts251 mutation can be phenotypically suppressed by mutations which map close to, or in, the gyrB gene and which affect the degree of DNA supercoillng. The level of suppression correlates with the degree of DNA relaxation observed, suggesting that the gene encoding the E. coli DNA ligase is activated by relaxation of the chromosomal DNA.
Molecular cloning and expression in Escherichia coli K‐12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci
Authors: Heuzenroeder M. W. , Beger D. W. , Thomas C. J. , Manning P. A. 
Published: October 2006

SummaryThe gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O‐antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM 1330expressed O‐antigen in E. coli K‐12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O‐antigen synthesis is genetically complex.Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O‐antigen biosynthesis in E. coli K‐12. Examination of LPS banding patterns of other O101 isolates by SDS‐PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was a close relationship among the 0101 ETEC isolates.
Protein G genes: structure and distribution of IgG‐binding and albumin‐binding domains
Authors: Sjöbring U. , Björck L. , Kastern W. 
Published: October 2006

SummaryProtein G (also designated Fc receptor type III) is the IgG‐binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG‐binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA‐hybridization experiments to study the structure and distribution of the albumin‐ and IgG‐binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG‐binding region (I) and the other corresponded to repeats in the albumin‐binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial species was found.
Fine structural analysis of the Zoogloea ramigera phbA‐phbB locus encoding β‐ketothiolase and acetoacetyl‐CoA reductase: nucleotide sequence of phbB
Authors: Peoples O. P. , Sinskey A. J. 
Published: October 2006

SummaryA series of expression plasmids containing either the complete insert from plasmid pUCDBKI (Peoples et al., 1987) or sub‐fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH‐specific acetoacetyl‐CoA reductase in the 2.3 kb of sequence located downstream from the β‐ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1 kb) of this region was determined. An open reading frame was located 88 bp downstream from the stop codon of the thiotase gene encoding a potential polypeptide of M, 25000, which is in good agreement with that observed for the overexpressed protein on SDS‐PAGE. N‐terminal protein sequence data obtained by Edman degradation of the purified Mr= 25000 polypeptide were used to identify the correct start of the NADPH‐specific acetoacetyl‐CoA reductase gene. Hence in Z. ramigera, the genes encoding β‐ketothiolase (phbA) and NADPH‐specific acetoacetyl‐CoA reductase (phbB) are organized as phbA‐phbB. S1‐nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.
Molecular characterization of a cell wall‐associated proteinase gene from Streptococcus lactis NCDO763
Authors: Kiwaki M. , Ikemura H. , Shimizu‐Kadota M. , Hiroshima A. 
Published: October 2006

Summary Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase‐positive phenotype (Prt). The 6.2kb HindIII‐Pstl fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the Pstl site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.
Sequence of the nagBACD operon in Escherichia coli K12 and pattern of transcription within the nag regulon
Authors: Plumbridge J. A. 
Published: October 2006

SummaryThe DNA sequence of a 3.6kb region downstream of the nagB gene (encoding glucosamJne‐6‐PO4‐deaminase) in Escherichia coli has been determined. Three open reading frames, which are subsequently referred to as nagA, nagC and nagD, were detected in this sequence. Genetic complementation and enzyme assays have shown that the first of these, nagA, encodes N‐acetyl glucosamine‐6‐phosphate deacetylase. Growth on N‐acetyl glucosamine induces the synthesis of a 1900 nucleotide long transcript which covers just nagE, encoding EIINag which is transcribed divergently from nagB, and of a 4200 nucleotide long transcript which covers all four ORFs of the nagB, A, C, D operon. More mRNA corresponding to nagB and nagA is detected than that corresponding to the distal genes, nagC and nagD. Considerable amounts of the induced mRNA are truncated molecules having their 3’ends after nagB and after nagA. Multiple 3′ RNA ends have been mapped after nagD and seem to correspond to the ends of transcripts stabilized by mRNA secondary structure (REP sequences) rather than transcription termination sites. A second promoter producing napD‐specific transcripts has been mapped just in front of the nagD gene.
DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus
Authors: Dorman C. J. , Lynch A. S. , Bhriain N. Ni , Higgins C. F. 
Published: October 2006

SummaryThe level of DNA supercoiling is crucial for many cellular processes, Including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toe and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.
Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus
Authors: Souillard N. , Sibold L. 
Published: October 2006

SummaryTwo regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen‐fixing archaebacterium Methanococcus thermolithotrophicus. A 2.8 kb Hindlll fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as 0RFnifH2. A 3.4kb Pstl fragment and an overlapping 3.B kb BglU fragment, containing the second region of homology, were cloned, and a DNA region of 4073bp was sequenced. It contained four complete open reading frames (ORFs) (ORFnifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORFnifH1‐ORF105‐ORF128‐ORFnifD‐ORFnifK. ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen‐fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions. A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment. A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1, This site is preceded, 21 bp upstream, by the sequence 5′‐TTTATATA‐3′ already found at the same position in several archaebacterial promoters. ORFnifH1 mRNA was too small to encode ORFnifDK. This was confirmed by the fact that another transcription start site was localized 85bp upstream from the ATG codon of ORFnifD.
Cloning and physical characterization of the L‐proline catabolism gene cluster of Aspergillus nidulans
Authors: Hull E. P. , Green P. M. , Arst H. N. , Scazzocchlo C. 
Published: October 2006

SummaryThe proline catabolism gene cluster of Aspergillus nidulans was cloned using a ‘brute force’ technique which detects clones hybridizing to restriction fragments overlapping chromosomal rearrangements. A number of deletion mutations and a translocation mutation in the cluster have been physically mapped, and an excellent correlation between the genetic and physical maps was established. Transcripts have been identified and orientated for each of the four genes of the cluster. All are monocistronic by size. All of the transcripts, including that of the regulatory gene prnA, are inducible. Using deletion endpoints and mRNA sizes, approximate gene positions on the physical map have been determined. Finally, the relationship between genetic and physical distance across the cluster has been estimated at 3‐4 kilobases per centiMorgan.
FNR‐dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR‐regulated gene expression
Authors: Spiro S. , Roberts R. E. , Guest J. R. 
Published: October 2006

SummaryThe ndh gene of Escherichia coli which encodes an NADH dehydrogenase contains a putative FNR‐binding site in its upstream non‐coding region, and its expression has been investigated using an ndh‐lacZ fusion. Expression of the fusion was found to be reduced during anaerobic growth, and experiments with hosts containing an fnr mutation and/or a multicopy fnr+ plasmid indicated that the anaerobic repression of the ndh gene is mediated by the FNR protein. Thus FNR can function as an anaerobic repressor as well as an anaerobic transcriptional activator. The results are consistent with the FNR‐binding function attributed to the proposed consensus sequence. Using frdA‐and ndh‐lacZ fusions exhibiting positive and negative regulation by FNR, it was further shown that the depletion of metal ions in growth media with chelating agents mimics oxygen with respect to the activity of FNR. Possible roles for metal ions in the oxygen‐sensing pathway associated with FNR function are discussed.
A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence‐associated antigens of Shigella flexneri
Authors: Adler B. , Sasakawa C. , Tobe T. , Makino S. , Komatsu K. , Yoshikawa M. 
Published: October 2006

SummaryThe expression of plasmid‐encoded, invasion‐related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence‐associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid‐encoded virulence genes.
Detection and identification of FaeC as a minor component of K88 fibriilae of Escherichia coli
Authors: Oudega B. , de Graaf M. , de Boer L. , Bakker D. , Vader C. E. M. , Mooi F. R. , de Graaf F. K. 
Published: October 2006

SummaryA tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit‐like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab‐encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.
Expression and phase variation of gonococcal P.11 genes in Escherichia coli involves ribosomal frameshifting and slipped‐strand mispairing
Authors: Belland R. J. , Morrison S. G. , van der Ley P. , Swanson J. 
Published: October 2006

SummaryExpression and phase variation of Neisseria gonorr‐hoeae P.ll genes in Escherichia coli viere studied using TnphoA fusions. Fusions were created in the P.llc gene of N. gonorrhoeae JS3 using λTnphoA‐1 and were characterized by restriction digestion and dideoxy sequencing. Three fusions were chosen for further study; Tnp7 (fusion junction at mature amino acid 7), Tnp57 (amino acid 57), Tnp66 (amino acid 66). All fusions were in frame with the P.llc coding sequence but were out of frame with the purported initiation codon. All fusion constructions were shown to phase vary in E. coli in an analogous fashion to that seen in N. gonorrhoeae, i.e. phase changes (in a recA background) at a frequency of c. 10−3 accompanied by an alteration at the DNA level of the number of coding repeats (CRs). In vitro mutagenesis of the fusion constructions indicated that expression of out of frame P.ll genes in E. coli was probably the result of ribosomal frameshifting within the run of ‘A’ residues immediately preceding the CR region and not due to low‐level false initiation at codons other than the ATG initiation codon (as had previously been suggested). The mechanism for P.llc::phoA phase variation appears to be related to the ‘slipped‐strand mispairing’ mechanism responsible for frameshift mutations in a number of other bacterial genes containing short, direct, tandem repeats.
Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities
Authors: Quinn F. D. , Tompkins L. S. 
Published: October 2006

SummaryThe DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using trans‐poson mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactrvation of proteolytic activity by trans‐poson insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200‐500bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.
Regulation of the phase switch controlling expression of type 1 fimbriae in Escherichia coli
Authors: Pallesen L. , Madsen O. , Klemm P. 
Published: October 2006

SummaryThe expression of Escherichia coli type 1 fimbriae is phase‐variable i.e. the bacterial cell is either fimbriated or non‐fimbriated. The transition from one state to the other is caused by the change in configuration of an invertible DNA segment harbouring the promoter of the fimA gene. The position of this phase switch is controlled by two proteins, FimB and FimE, which mediate an ‘on’ or ‘off’ configuration of the switch, respectively. In this study, we have investigated how these proteins control the switch by means of fim‐lac fusions on low‐copy‐number plasmids. It was found, by in trans and cis complementation, that the ratio of fimB to fimE and the total concentration of the gene products determine the configuration of the switch as well as the frequency of phase switching. It was also shown that transcription occurs from the promoter located at the phase switch when this is in the ‘off’ configuration. This suggests a regulatory mechanism, since the resulting transcript would be anti‐sense to the fimE transcript.
Specificity of signal molecules in the activation of Agrobacterium virulence gene expression
Authors: Melchers L. S. , Regensburg‐Tuïnk A. J. G. , Schilperoort R. A. , Hooykaas P. J. J. 
Published: October 2006

SummaryThe activation of the Agrobacterium virulence system is known to be induced by certain phenolic compounds. We have tested the vir‐inducing ability of fifty compounds, by using a virB‐lacZ gene fusion, and analysed the relationship between structure and activity of these compounds. In this way we have identified several new vir‐inducers: coniferylalcohol, 3,5‐dimethoxy‐4‐hydroxybenzene, homovanillic acid, ferulic acid, 3‐ethoxy‐4‐hydroxybenzaldehyde and guaiacol, all of which are compounds with strong or moderate activity and four compounds with weak vir‐inducing activity. In view of the specificity of vir‐inducers, our data extended observations of others and enabled us to define the specific structural features of a vir‐inducer molecule. In addition we show here that induction of the octopine Ti vir‐genes is (i) optimal at 29° C and totally abolished at 37° C., and (ii) strongly inhibited at low concentrations of sodium chloride. The implications for plant transformation are discussed.
Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin
Authors: Niven D. F. , Donga J. , Archibald F. S. 
Published: October 2006

SummaryOuter membranes from Haemophilus pleuropneumoniae grown under iron‐replete and iron‐restricted conditions in vitro were analysed by means of SDS‐PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96–102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropnuemoniae during‘growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron‐restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron‐regulated outer membrane protein.
Signalling proteins in enterobacterial AmpC β‐lactamase regulation
Authors: Lindquist S. , Galleni M. , Lindberg F. , Normark S. 
Published: October 2006

SummaryThe cloned Citrobacter freundii ampC β‐lactamase is inducible in the presence of its regulatory gene ampR in Escherichia coli (Lindberg et al., 1985). The basal level of expression and inducibility are affected by two E. coli proteins encoded by the closely linked ampD and ampE genes. Deletion of both genes led to constitutive ampR‐dependent overproduction of β‐lactamase, whereas an out‐of‐frame deletion in AmpD caused the basal expression to increase twofold. This ampD1 mutant was inducible at lower β‐lactam concentrations than the wild type. An IS1 insertion in ampD was polar on ampE expression and increased basal β‐lactamase expression 30‐fold while mediating a semi‐constitutive phenotype. AmpE expressed from a recombinant plasmid in an ampD ampE deletion mutant reduced basal β‐lactamase expression to wild‐type levels but did not markedly reduce β‐lactam resistance since the cells became hyperinducible. in the absence of AmpD, increasing levels of AmpE therefore decrease the basal expression of AmpC β‐lactamase in an AmpR‐dependent manner. AmpD modulated the response exerted on β‐lactamase expression by AmpE. The ampD gene encodes a 20.5kD cytoplasmic protein while the 32.1kD ampE gene product is an integral membrane protein with a likely ATP‐binding site between the second and third putative transmembrane region. Since neither AmpD nor AmpE are needed for β‐lactam induction and since these proteins could not be covalently labelled by benzylpenicillin, they are not thought to act as β‐lactam‐binding sensory tranducers. Instead it is suggested that AmpD and AmpE sense the effect of β‐lactam action on peptidoglycan biosynthesis and relay this signal to AmpR.