bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

Protein 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

A 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

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

The 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

The 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

Two 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

The 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

The 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

The 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

A 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

Expression 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

The 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

The 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

The 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

Outer 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

The 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.
Regulation of enterobacterial cephalosporinase production: the role of a membrane‐bound sensory transducer
Authors: Honoré N. , Nicolas M.‐H. , Cole S.T.
Published: October 2006

In clinical isolates of Enterobacter cloacae, resistance to the newer β‐lactam antibiotics often results from overproduction of a cephalosporinase encoded by the β‐lactam‐inducible ampC gene. Regulation of ampC is controlled by the divergently expressed activator gene, ampR, and a second unlinked locus. In this presentation we show that although Escherichia coli has IQst its ampR gene it has retained the second regulatory locus and that this comprises the bicistronic ampDE operon. Genetic and biochemical studies define the ampD gene as encoding a repressor for amp C transcription whereas the ampE gene product is a cytoplasmic membrane protein. Inactivation of the AmpD protein by mutation causes massive overproduction of cephalosporinase which, in E. cloacae, can terminate in therapeutic failure. In contrast, toss of AmpE results in a total block in induction, despite the presence of the activator, AmpR.
Functional organization and nucleotide sequence of virulence Region‐2 on the large virulence plasmid in Shigella flexneri 2a
Authors: Sasakawa C. , Adler B. , Tobe T. , Okada N. , Nagai S. , Komatsu K. , Yoshikawa M.
Published: October 2006

The 7kb virulence Region‐2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS‐polyacrylamide gel electro‐phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)‐kiloDalton (kD) proteins. Complementation of Tn5‐induced mutations in Region‐2 with the above plasmid constructs indicated that Region‐2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region‐2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full‐length RNA of 7.5 kb which covers Region‐2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region‐2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.
Conserved serine‐rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing
Authors: Hall J. , Hazlewood G. P. , Huskisson N. S. , Durrant A. J. , Gilbert H. J.
Published: October 2006

The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N‐terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N‐terminal signal peptide had been deleted, still expressed an enzyme which was secreted in Escherichia coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine‐rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C‐rich region of dyad symmetry (δG = 24kcal) characteristic of E. coli Rho‐independent transcription terminators.
Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin
Authors: Donohue‐Rolfe A. , Jacewicz M. , Keusch G. T.
Published: October 2006

Shiga toxin is a potent protein toxin produced by Shigella dysenteriae type I strains. In this report we present a procedure for the separation of functionally intact toxin A and B chains and for their reconstitution to form biologically active molecules. In agreement with the findings of others, the isolated A chain was shown to be a potent in vitro inhibitor of eukaryotic protein synthesis. The isolated B chain bound to HeLa cells and competitively inhibited the binding and cyto‐toxic activity of holotoxin. These findings show that the functional role of the B chain is to recognize cell surface functional receptors. By labelling the B sub‐unit alone, prior to renaturatlon of holotoxin, the polypeptide chains were shown to associate non‐covalently with a stoichiometry of one A chain and five B chains.
The role of the sporulation gene spollIE in the regulation of prespore‐specific gene expression in Bacillus subtilis
Authors: Foulger D. , Errington J.
Published: October 2006

The spollIG gene encodes a sigma factor that determines prespore‐specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spollIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spollIE, has a specific effect on spollIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spollIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore‐specific gene expression in this system.
Osmotic regulation of porin expression: a role for DNA supercoiling
Authors: Graeme‐Cook K. A. , May G. , Bremer E. , Higgins C. F.
Published: October 2006

The OmpC and OmpF porins are major outer membrane proteins of Escherichia coli and Saimonella typhimurium. Their expression is affected by many environmental factors and by mutations in a variety of independent genes. The pair of regulatory proteins, OmpR and EnvZ, are required for normal porin expression. Despite intensive investigation, the mechanisms by which porin expression is regulated remain unclear. Mutations which alter supercoiling, as well as inhibitors of DNA gyrase, show that porin expression is extremely and specifically sensitive to the level of DNA supercoiling. Our data lead us to suggest that environmentally induced changes in DNA supercoiling may play a role in determining the level of porin expression. These findings have implications for current models of porin regulation.
Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70 kD heat‐shock protein family
Authors: Mehlert A. , Young D. B.
Published: October 2006

A 71 kiloDalton antigen from Mycobacterium tuberculosis is recognized by antibodies and by T lymphocytes during infection (Britton et al., 1986a). Partial sequence analysis indicates a relationship between this antigen and the highly conserved family of 70‐kiloDalton heat shock proteins (hsp70) (Young et al., 1988). Biochemical and serological characterization of the protein confirms its membership of the hsp70 gene family, and metabolic labelling demonstrates that it is a major component of the mycobacterial response to heat stress. The role of stress proteins as antigens during infection is discussed.
The streptokinase gene of group A streptococci: cloning, expression in Escherichia coli, and sequence analysis
Authors: Huang T.‐T. , Malke H. , Ferretti J. J.
Published: October 2006

The gene specifying the group A streptokinase (ska) gene was cloned from an M type 49 strain of Streptococcus pyogenes and shown to express in Escherichia coli. The nucleotide sequence of the DNA fragment carrying ska was determined and compared to that of the group C streptokinase gene (skc). There is 90% sequence identity between the two genes, with highly conserved transcription and translation control regions. The deduced amino acid sequence of the group A streptokinase (SKA) contains the same number of amino acids as that of group C streptokinase, with 85% sequence identity between the two proteins. Among 440 amino acid residues specified by the coding sequence, there are 62 non‐identical residues with 45 conserved and 17 non‐conserved residues. The non‐identical residues are located in two major regions, spanning residues 174 to 244 and 270 to 290, with 40 and 10 amino acid changes, respectively. The sequence differences provide an explanation at the molecular level for the previous findings of immunological and chemical heterogeneity among streptokinases produced by pathogenic streptococci.
Molecular evolution of ubiquitous β‐lactamases towards extended‐spectrum enzymes active against newer β‐lactam antibiotics
Authors: Collatz E. , Labia R. , Gutmann L.
Published: October 2006

Production of β‐lactamases, and of the plasmid‐encoded TEM‐and SHV‐type enzymes in particular, is the most common mechanism of resistance against β‐lactam antibiotics in Gram‐negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first‐and second‐generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally ‘β‐lactamase‐stable’ third‐generation cephalosporins or by mono‐bactams, which fall into the substrate range of the mutant or ‘extended‐spectrum’β‐lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the α‐3 helix), 162 (adjacent to the α‐7 helix) and 235,236 and 237 (on the β‐3 strand) are located in close proximity to the active‐site cavity and are thought to open up novel enzyme‐substrate interactions, involving, in particular, the oxyimino moieties of the newer β‐lactam compounds.
The molecular structure of the Corynebacterium glutamicum threonine synthase gene
Authors: Han K.‐S. , Archer J. A. C. , Sinskey A. J.
Published: October 2006

The minimal region encoding the Corynebacterium glutamicum threonine synthase structural gene and its promoter was mapped by deletion analysis and complementation of the C. glutamicum thrC allele to a 1.6kb region of the recombinant plasmid pFS80. The nucleotide sequence of this and flanking DNA was determined. The transcription and translation start points were identified by S1 mapping analysis and amino‐terminal protein sequencing, respectively. The thrC gene encodes a 54481‐Dalton polypeptide product. Translation of the thrC mRNA initiates only six nucleotides downstream from transcription. The length of the mRNA transcript is consistent with a single gene transcription unit. The C. glutamicum thrC gene is expressed independently of the other threonine‐specific genes hom and thrB.
Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences?
Authors: Fayet O. , Ramond P. , Polard P. , Prère M. F. , Chandler M.
Published: October 2006

Members of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame In the –1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the Integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a −1 frameshift resulting in the production of a fusion protein.
Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia Coli
Authors: Klaasen P. , de Graaf F. K.
Published: October 2006

Expression of the 987P gene cluster is activated by the adjacent IS1 element of an STpa transposon. Nucleotide sequence analysis of the 987P‐DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30349 Daltons. The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY. Moreover, a 43‐amino‐acid residue sequence in the C‐terminal part of FapR is similar to the C‐terminal domain of AraC, RhaR, and RhaS. Expression of fapR is dependent on the adjacent IS1 element. The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC
Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens
Authors: Nichols W. A. , Clegg S. , Brown M. R.
Published: October 2006

The nucleotide sequence of a DNA fragment that contains the fimA gene, encoding the major fimbrial subunit, of Serratia marcescens IA506 and associated flanking sequences has been elucidated. In addition, the origin of transcription has been identified and is located 120 base pairs upstream of the fimA initiation codon. The predicted amino acid sequence of the FimA polypeptide exhibits some degree of sequence homology with the fimbrial subunits encoded by the fimA determinants of Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli and also to Smf2, the major structural component of mannose‐resistant (MR) fimbriae of S. marcescens. The Serratia adhesin that facilitates haemagglutination mediated by type 1 fimbriae is less susceptible to inhibition by D‐mannose than has been observed to be the case in other type 1 fimbrial adhesins. The molecule conferring this adherence specificity has been shown to be distinct from the fimA gene product and, therefore, is analogous to the fimbrial systems reported in other species.
Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis
Authors: Lövgren A. , Zhang M. , Engström A. , Dalhammar G. , Landén R.
Published: October 2006

The gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA‐related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 ± 2.5 ng per mg of larval body weight.
Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae
Authors: Au‐Young J. , Robbins P. W.
Published: October 2006

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH‐activity profiles showed that yeast and hyphae contain a protease‐dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin syntheses in C albicans.

A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane‐bound zymogen capable of chitin biosynthesis in vitro was based on several criteria, (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]‐GlcNAc from the substrate, UDP[U‐14C]‐GlcNAc, into alkali‐insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH‐activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 6.0 is proposed.

DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).
Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenylase components
Authors: Böhm R. , Sauter M. , Böck A.
Published: October 2006

An 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (0RF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron‐sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH‐ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.
Two precursors of the heat‐stable enterotoxin of Escherichia coli: evidence of extracellular processing
Authors: Rasheed J. K. , Guzmán‐Verduzco L.‐M. , Kupersztoch Y. M.
Published: October 2006

Expression of the gene of the methanol‐soluble, heat‐stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS‐PAGE of a cell‐associated 7500 Dalton STA‐related peptide; when similar experiments were performed with a phosphate buffer SDS‐PAGE system, an additional Mr9800 band became apparent. The 9800 Dalton form, pre‐pro‐STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m‐chlorophenylhydrazone); by pulse‐chase experiments, it was shown that pre‐pro‐STA became a periplasmic Mr 7500 pro‐STA and this form was chased to the culture supernatant; periplasmic and extracellular pro‐STA showed the same electrophoretic mobility. A short time after the pulse, pro‐STA was converted extracellularly to mature STA (Mr 4500). It is proposed that STA is synthesized as pre‐pro‐STA, a 72‐amino‐acid peptide that is subsequently cleaved between amino acids 19 and 20as it is translocated across the inner membrane. The resulting 53‐amino‐acid pro‐STA is first detected in the periplasm and is then secreted to the culture supernatant. Pro‐STA is cleaved extracellularly to yield mature STA (Mr 4500).
A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils
Authors: Stinavage P. S. , Martin L. E. , Spitznagel J. K.
Published: October 2006

We have isolated a Salmonella typhimurium (ST) mutant, JKS400, deficient in the production of a surface‐exposed outer membrane protein (Omp) and phenotypically hypersensitive to the oxidative antimicrobial mechanism of polymorphonuclear leukocytes (PMNs). This Omp migrated at approximately 59 kiloDaltons (kD) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). We found with P22 transduction that the capacities to produce the protein and to exert wild‐type resistance to oxidative killing were tightly linked.

Transduction of JKS400 with a P22(HT)Ht bacteriophage grown on a Tn10 insertion library in LT2 yielded tetracycline‐resistant isolates that had been returned to wild‐type protein production. Further experiments showed that restoration of protein production was accompanied by restoration of the parental resistance phenotype to killing by PMNs and by restoration to wild‐type resistance to H2O2. The map position of the Tn10 was determined to be at 96 minutes in the Salmonella chromosome.

This protein appears to behave as a virulence factor, promoting the capacity of Salmonella typhimurium LT2 to survive oxygen‐dependent killing mechanisms in neutrophils.
Effect of positive redox potentials (>+400mV) on the expression of anaerobic respiratory enzymes in Escherichia coli
Authors: Unden G. , Trageser M. , Duchêne A.
Published: October 2006

The expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (E>h) in the medium. Redox potentials up to +300mV allowed full expression of fumarate reductase (frd) genes. Higher values resulted in decreased expression. The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential E0′=+400mV to 440 mV. At Eh values >+510mV (generated anaerobically by hexacyanoferrate(III)) the degree of repression was the same as that obtained by O2. Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases. Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of E0′=+400mV to 440mV.
Aging and senescence of the budding yeast Saccharomyces cerevisiae
Authors: Jazwinski S. M.
Published: October 2006

The budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides. Longevity in this organism involves a genetic component. Several morphological and physiological changes are associated with yeast aging and senescence. One of these, an increase in generation time with age, provides a ‘biomarker’ for the aging process. This increase in generation time has revealed the operation of a ‘ senescence factor(s)’, which is likely to be a product of age‐specific gene expression. The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span. It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.
Sequence of the fhuE outer‐membrane receptor gene of Escherichia coli K12 and properties of mutants
Authors: Sauer M. , Hantke K. , Braun V.
Published: October 2006

The fhuE gene of Escherichia coli codes for an outer‐membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77453, which agrees with the molecular weight of 76000 determined by poly‐acrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB‐dependent receptors. A valine to proline exchange in the ‘TonB box’ absolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited Increasing iron‐coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C‐terminal end was exported to the periplasm but failed to be inserted into the outer membrane.
The regulatory role of the IS 1‐encoded InsA protein in transposition
Authors: Zerbib D. , Polard P. , Escoubas J. M. , Galas D. , Chandler M.
Published: October 2006

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1‐mediated cointegrate formation and transposition of a synthetic IS 1‐based transposon (‘omegon’Ω‐on). These results also indicate that the Ω‐on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA‐lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1‐encoded genes both in cis or in trans. The experiments involving Ω‐on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.