bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP‐ and NarL‐dependent regulation
Authors: Tyson K. L. , Cole J. A. , Busby S. J. W.
Published: October 2006

Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the ‐10 hexamer and the FNR‐binding site, and to find the sites essential for nitrite‐dependent activation and nitrate‐dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL‐dependent repression by nitrate is due to two heptamer sequences that flank the FNR‐binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.
Transcription of the principal sigma‐factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase
Authors: Fujita Masaya , Tanaka Kan , Takahashi Hideo , Amemural Akinori
Published: October 2006

The rpoS gene encodes the second principal sigma factor of RNA polymerase in stationary‐phase cells in Escherichia coli. We examined the transcription of Pseudomonas aeruginosa rpoS as to the growth of ceils. The results of quantitative S1 nuclease mapping of rpoS and rpoD, encoding the principal sigma factor, indicated that the transcription of rpoS is induced in stationary‐phase cells, whereas that of rpoD is induced in exponential‐phase cells. By high‐resolution S1 nuclease mapping, the 5′‐ and 3′‐ends of rpoS mRNA were determined. The results indicated that rpoS is transcribed as a monocistronic mRNA. The sequence preceding the 5 end of rpoS mRNA showed poor homology to the consensus sequences of the previously known promoters. P. aeruginosa rpoS was not transcribed in E coli. By in vitro transcription assaying, P. aeruginosa rpoS was shown to be transcribed by the RNA polymerase fraction containing the principal sigma (σ70) RNA polymerase of P. aeruginosa
Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis
Authors: Kado Clarence I.
Published: October 2006

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram‐negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T‐DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity=50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase‐I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase‐I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptl operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA‐protein complexes.
A mutation in the receiver domain of the Agrobacterium tumefaciens transcriptional regulator VirG increases its affinity for operator DNA
Authors: Han Dong Cho , Winans Stephen C.
Published: October 2006

We fused the wild‐type Agrobacterium tumefaciens virG gene and the constitutive virGN54D allele to the malE gene of Escherichia coli, and studied the binding of MBP—VirG fusions to the autoregulated virG promoter. MBP—VirGN54D protein bound this promoter with 10‐fold higher affinity than MBP—VirG, and bound to vir box I with eightfold higher affinity than to vir box III. Disruption of vir box III did not alter the affinity for vir box I, suggesting a lack of cooperativity between these sites. We provide evidence that protein bound at a single vir box may have a higher oligomeric state than non‐bound protein, and that a DNA distortion adjacent to vir box I may occur during activation.
Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome‐binding site permits tap‐independent RepA synthesis
Authors: Blomberg Pontus , Engdahl Hilde M. , Malmgren Charlotta , Romby Pascale , Wagner E. Gerhart H.
Published: October 2006

The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post‐transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem‐loop sequestering the repA ribosome‐binding site blocks tap translation‐independent repA expression. Destabilization of this structure resulted in tap‐independent RepA synthesis, concomitant with a loss of CopA‐mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome‐binding site stem‐loop. An increased spacing between the repA Shine‐Dalgarno region and the start codon permitted even higher rep A expression. In Incα/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.
Structural and functional diversity among bacterial interspersed mosaic elements (BIMEs)
Authors: Bachellier Sophie , Saurin William , Perrin David , Hofnung Maurice , Gilson Eric
Published: October 2006

Palindromic units (PU or REP) were defined as 40‐nucleotide DNA sequences which are highly repeated in the genome of several members of the Enterobacteriaceae. They were shown to be a constituent of the bacterial interspersed mosaic element (BIME), in which they are associated with other repetitive sequences. We report here that Escherichia coli PU sequences contain three motifs (Y, Z1 and Z2), leading to the definition of two BIME families. The BIME‐1 family, highly conserved over 145 nucleotides, contains two PUs (motifs Y and Z1). The BIME‐2 family contains a variable number of PUs (motifs Y and Z2). We present evidence, using band shift experiments, that each PU motif binds DNA gyrase with a different affinity. This suggests that the two families are functionally distinct.
Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli
Authors: Girón Jorge A. , Levine Myron M. , Kaper James B.
Published: October 2006

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera‐like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH2‐terminal amino acid sequence revealed homology with the toxin‐coregulated pilus of Vibrio cholerae, the bundle‐forming pilus of enteropathogenic E. coli and type IV pilins of some Gram‐negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K‐12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno‐prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat‐stable toxin only.
Expression of the nmpC gene of Escherichia coli K‐12 is modulated by external pH. Identification of cis‐acting regulatory sequences involved in this regulation
Authors: Coll J. L. , Heyde M. , Portalier R.
Published: October 2006

Using a set of gene fusions generated with TnphoA, we previously identified the phmA locus, whose expression is modulated as a function of external pH (pHo). The phmA::phoA fusion was cloned and sequenced and the phmA locus was identified with the nmpC gene. This gene lies within the defective lambdoid prophage qsr′ and NmpC is an outer membrane protein which functions as a porin. We demonstrated that nmpC is sensitive to catabotite repression and dependent on the CRP—cAMP complex. However, cAMP is not a signal for the pHo‐dependent expression of nmpC. By generating step deletions in the sequence 5′ to the nmpC coding region, we identified a DNA region in position —345 to —127 which is involved in nmpC repression, mainly during growth at acid pHo. Four regions with strong homologies and a very well‐conserved organization of the functional sequence were found in the nmpC and ompF promoters. We propose that the negative regulation of nmpC during growth at low pHo might involve DNA looping of the nmpC promoter.
The pdhR–aceEF–lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex
Authors: Quail M. A. , Haydon D. J. , Guest J. R.
Published: October 2006

Transcript mapping and studies with lacZ translational fusions have shown that the pdhR gene (formerly genA) is the proximal gene of the pdhR–aceE–aceF–Ipd operon encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli. A pdhR–lpd read‐through transcript (7.4 kb) initiating at the pyruvate‐inducible pdh promoter, and a smaller lpd transcript (1.7 kb) initiating at the independent lpd promoter, were identified. Evidence showing that the pdhR gene product negatively regulates the synthesis of the PdhR protein and the PDH complex via the pdh promoter was obtained, with pyruvate (or a derivative) serving as the putative inducing coeffector. The partially purified PdhR protein was also found to specifically retard and protect DNA fragments containing the pdh promoter region. The pdh promoter was not strongly controlled by ArcA, FNR or CRP.
Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase
Authors: Palzkill Timothy , Le Quyen‐Quyen , Venkatachalam K. V. , LaRocco Mark , Ocera Hermes
Published: October 2006

In order to understand how TEM‐1 β‐lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161–170 region includes a portion of an omega loop structure, which is involved in the formation of the active‐site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended‐spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild‐type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161‐170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100‐fold increase in ceftazidime resistance were identified. Characterization of altered β‐lactamase enzymes indicated that while their catalytic efficiency (Kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild‐type TEM‐1 β‐lactamase.
Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H‐NS
Authors: Zuber F. , Kotlarz D. , Rimsky S. , Buc H.
Published: October 2006

Replacement of the CRP‐binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H‐NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H‐NS may repress promoters both at short and long distances from a favoured binding site are discussed.
The porA gene in serogroup A meningococci: evolutionary stability and mechanism of genetic variation
Authors: Suker Janet , Feavers Ian M. , Achtman Mark , Morelli Giovanna , Wang Jian‐Fu , Maiden Martin C.J.
Published: October 2006

Molecular analyses were applied to the genes encoding variants of the serosubtyping antigen, the class 1outer membrane protein (PorA), from 55 serogroup A Neisseria meningitidis strains. These genes were evolutionarily stable and exhibited a limited range of genetic variation, primarily generated by recombination. Translation of the gene sequences revealed a total of 19 distinct amino acid sequences in the variable regions of the protein, 6 of which were not recognized by currently available serosubtyping monoclonal antibodies. Knowledge of these aminoacid sequences permitted a rational re‐assignment of serosubtype names. Comparison of the complete genes with porA gene sequences from serogroup B and C meningococci showed that serogroup A possessed a limited number of the possible porA genes from a globally distributed gene pool. Each serogroup A subgroup was characterized by one of four porA gene types, probably acquired upon subgroup divergence, which was stable over periods of decades and during epidemiological spread. Comparison with other variable genes (pil and iga) indicated that the three alleles were independently assorted within the subgroup, suggesting that their gene types were older than the subgroups in which they occurred.
Deregulation of temperature‐dependent transcription of the invasion regulatory gene, virB, in Shigella by rho mutation
Authors: Tobe Toru , Yoshikawa Masanosuke , Sasakawa Chihiro
Published: October 2006

Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature‐regulated. virB transcription is under the control of VirF and H‐NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30° C and 37° C was isolated using miniTn10‐Kan (mini KAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C‐terminus of the Rho protein. Consequently, decreased transcription termination activity at a p‐dependent terminator, λtL1, was observed, in the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30°C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoler, since the effects of the mutation on virB transcription were abolished when its promoler region was replaced by the tac promoler. Temperature‐responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30° C as well as at 37°C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature‐dependent changes in DNA structure, thus contributing to thermoregulation of VirS transcription.
Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein
Authors: Schmitt Wolfgang , Haas Rainer
Published: October 2006

The human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac+), all of which produce the 87kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac−) also carry the gene, Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33‐amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C‐termlnal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease‐type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.
DNA‐binding proteins as site‐specific nucleases
Authors: Pan Clark Q. , Landgraf Rail , Sigman David S.
Published: October 2006

DNA‐binding proteins can be converted into site‐specific nucleases by linking them to the chemical nuclease 1,10‐phenanthroline‐copper. This can be readily accomplished by converting a minor groove‐proximal amino acid to a cysteine residue using site‐directed mutagenesis and then chemically modifying the sulphydryl group with 5‐iodoacetamido‐1,10‐ phenanthroline‐copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of proteins within genomes.
Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibres
Authors: Sandmeler Heinrich
Published: October 2006

Molecular analysis reveals a surprising sharing of short gene segments among a variety of large double‐stranded DNA bacteriophages of enteric bacteria. Ancestral genomes from otherwise unrelated phages, including λ Mu, P1, P2 and T4, must have exchanged parts of their tail‐fibre genes, Individual genes appear as mosaics with parts derived from a common gene pool. Therefore, horizontal gene transfer emerges as a major factor in the evolution of a specific part of phage genomes. Current concepts of homologous recombination cannot account for the formation of such chimeric genes and the recombinational mechanisms responsible are not known. However, recombination sites for DNA invertases and recombination site‐like sequences are present at the boundaries of gene segments conferring the specificity for the host receptor. This, together with the properties of the DNA inversion mechanism, suggests that these site‐specific recombination enzymes could be responsible for the exchange of host‐range determinants.
CooC and CooD are required for assembly of CS1 pili
Authors: Froehlich Barbara J. , Karakashian Alexander , Meisen Lawrence R. , Wakefield Jeffrey C. , Scott June R.
Published: October 2006

Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple Identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD Is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K‐12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K‐12 and constructed in an ETEC‐derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.
Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β‐1,4‐exoceilobiohydrolase analogous to Trichoderma reesei CBH II
Authors: Meinke Andreas , Gilkes Neil R. , Kwan Emily , Kilburn Douglas G. , Warren R. Antony J. , Miller Robert C.
Published: October 2006

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N‐terminal catalytic domain, three repeated sequences of 95 amino acids, and a C‐terminal cellulose‐binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.
Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) ‘minicircle’: roles of open reading frames and origin of tandem insertions
Authors: Smokvina Tamara , Henderson Duncan J. , Melton Rachel E. , Brolle Dirk‐F , Kieser Tobias , Hopwood David A.
Published: October 2006

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20‐fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild‐type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co‐transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.
Regulation of the xylanase‐encoding xlnA gene of Aspergilius tubigensis
Authors: de Graaff Leo K. , van den Broeck Hetty C. , van Ooijen Albert J. J. , Visser Jaap
Published: October 2006

A gene encoding an endo‐1,4‐β‐xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5′ upstream region of the gene a 158bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase‐encoding gene. From the results with this reporter system it is concluded that this 158bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xinA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. niduians. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in ail three aspergilli.
Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitumβ‐lactamase genes cloned from a natural isolate and a high‐level β‐lactamase producer
Authors: Timm J. , Perilli M. G. , Duez C. , Trias J. , Orefici G. , Fattorini L. , Amicosante G. , Oratore A. , Joris B. , Frére J. M. , Pugsley A. P. , Gicquel B.
Published: October 2006

The gene encoding a class A β‐lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high‐level amoxicillin‐resistant mutant that produces large amounts of β‐lactamase (blaF). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high‐level production of the β‐lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N‐terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or biaF) to the E. coli gene phoA resulted in the production of BlaF‐PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.
A two‐component signal‐transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae
Authors: Guenzi Eric , Gasc Anne‐Marie , Sicard Michel A. , Hakenbeck Regine
Published: October 2006

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin‐binding protein (PBP) variants with decreased affinities for β‐lactam antibiotics. Cefotaxime‐resistant laboratory mutants, selected after several steps on increasing concentrations of this β‐lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal‐transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N‐terminal extracelluiar sensor part, and an intracelluiar C‐terminal domain with the conserved His‐226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime‐resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime‐resistant mutants were each located in the C‐terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal‐transducing system cia is involved in early steps of competence regulation.
Frequency of deletion formation decreases exponentially with distance between short direct repeats
Authors: Chédin F. , Dervyn E. , Dervyn R. , Ehrlich S. D. , Noirot P.
Published: October 2006

The effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000‐fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA‐duplex flexibility. A similar relationship between deletion formation and distance was observed in a θ‐replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.
Role of rpoS (katF) in oxyR‐independent regulation of hydroperoxidase I in Escherichia coli
Authors: Ivanova Anna , Miller Cathy , Glinsky Gennadi , Eisenstark Abraham
Published: October 2006

We present evidence showing that rpoS (katF) is a regulator of katG gene transcription In an oxyR‐independent manner. Mutation of the rpoS gene in several different Escherichia coli strains caused a significant reduction in catalase HPI activity. In rpoSδoxyR double mutants, the level of HPI was considerably lower compared to the δoxyR parent strain, and was restored when transformed with an rpoS+ plasmid. Overproduction of HPI in oxyR suppressor strains was greatly diminished after inactivation of the rpoS gene and was accompanied by a substantial increase in sensitivity to menadione. Beta‐galactostdase expression from a katG::lacZ promoter was lower in rpoS strains compared to rpoS+ isogenic parents. Several δoxyR strains had detectable levels of katG transcription that was significantly diminished after rpoS gene inactivation.
Identification of lactoferrin‐binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola
Authors: Staggs Teanna M. , Greer Margaret K. , Baseman Joel B. , Holt Stanley C. , Tryon Victor V.
Published: October 2006

Lactoferrin‐binding or ‐associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent‐solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29‐34 kDa exhibited association with human apo‐ and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin‐associated proteins from T. pallidum and T. denticola in competitive‐binding assays. However, the T. denticola proteins dissociated from a lacto‐ferrin‐affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29‐34 kDa band of T. pallidum suggesting that the polypeptide was lipid‐modified. Each of the lactoferrin‐binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin‐binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.
‘Muprints’ of the lac operon demonstrate physiological control over the randomness of in vivo transposition
Authors: Wang Xiuhua , Higgins N. Patrick
Published: October 2006

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments In Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O1 and O2 operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes In transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O2 operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non‐expressed genes, Mu may demonstrate a self‐restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.
Protein folding in the periplasm of Escherichia coli
Authors: Wülfing Christoph , Plückthun Andreas
Published: October 2006

With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right. Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E. coli is very recent and is often speculative. This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E. coli proteins involved in this process. After an introduction to the specific physiological situation in the periplasm of E. coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation — disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones — and briefly cover the guided assembly of multi‐subunit structures. Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena.
A group A streptococcal Enn protein potentially resulting from intergenomic recombination exhibits atypical immunoglobulin‐binding characteristics
Authors: Podblelski A. , Hawlitzky J. , Pack T. D. , Flosdorff A. , Boyle M. D. P.
Published: October 2006

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA‐binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG3. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C‐terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N‐terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M‐like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N‐terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N‐terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG3. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig‐binding characteristics.
Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens
Authors: Lyristis Michael , Bryant Amy E. , Sloan Joan , Awad Milena M. , Nisbet Ian T. , Stevens Dennis L. , Rood Julian I.
Published: October 2006

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild‐type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916‐derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild‐type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two‐component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane‐spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two‐component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.
Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis
Authors: Abe A. , Matsui H. , Danbara H. , Tanaka K. , Takahashi H. , Kawahara K.
Published: October 2006

The expression regulation of spvR, a regulatory gene on the virulence plasmid (pKDSC50) of Salmonella choleraesuis serovar Choleraesuis, was investigated by spvR–lacZ translational fusion. The spvR gene was found to be positively regulated by its own product, the SpvR protein, and this unusual positive auto‐regulation was repressed by the products of spvA and spvB, virulence‐associated genes present downstream from the spvR gene. Amino acid sequence analysis revealed that the amino‐terminal region of SpvB had homology with the CatM repressor protein of Acineto‐bacter calcoaceticus, which belongs to the MetR/LysR protein family. On the other hand, the sigma factor RpoS was required for expression of the spvR gene in the stationary phase of bacterial growth. The SpvR protein was also necessary for self‐activation, suggesting that an RNA polymerase holoenzyme containing RpoS requires SpvR protein in order to recognize the spvR promoter.
Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion‐defective mutants
Authors: Kahrs Andreas F. , Bihlmater Armin , Facius Dirk , Meyer Thomas F.
Published: October 2006

One requirement for the invasion of, and tight adherence to, human epitheiial cells by Neisseria gonorrhoeae is the synthesis of distinct opacity (Opa) outer membrane proteins, encoded by a family of phase‐variable chromosomal genes. However, cloning and surface expression of invasion‐promoting Opas in Escherichia coli is not sufficient for the efficient invasion of epithelial cells: additional factors besides Opa may be involved in this process. Using the phoA mini‐transposon TnMax4, a library of gonococcal mutants affected in the expression of genes encoding exported proteins was generated through shuttle mutagenesis. Of a total of 608 PhoA+ plasmid clones identified in E. coli E145 approximately 40% were used successfully in transforming N. gonorrhoeae and in activating the corresponding chromosomal genes. Gonococci producing the invasion‐promoting Opa50 served as the genetic background to identify 51 mutants unable to enter Chang human epithelial cells. We expect some of these mutations affect the interaction of N. gonorrhoeae with epithelial cells directly, while other mutants may carry defects in general house‐keeping, secretory and/or regulatory determinants. In some mutants the loss of invasiveness appears to be due to a negative dominant effect of the PhoA+ fusions produced in these mutants. Some of the identified genes display a phase‐variation phenomenon in E. coli and several genes are found in multiple copies in N. gonorrhoeae and/or present only in pathogenic Neisseria species.
Streptococcus pyogenes type IIa IgG Fc receptor expression is co‐ordinately regulated with M protein and streptococcal C5a peptidase
Authors: La Penta Diqui , Zhang Xin Pin , Cleary P. Patrick
Published: October 2006

Streptococcus pyogenes is an important agent of human disease which expresses a variety of proteins and polysaccharides on its surface. Surface molecules M protein and streptococcal C5a peptidase (SCPA) are virulence factors which undergo concurrent phase variation and are under the co‐ordinate control of the virR locus. Most opacity factor‐positive (OF+) strains of S. pyogenes also express IgG Fc receptor proteins on their surface. These studies were initiated to determine whether the type IIa Fc receptor on the surface of S. pyogenes phase‐varies with members of this regulatory circuit. Several methods were applied to M− and M− variant strains to evaluate this question, (i) Immunoblot assays quantified Fc receptors on whole cells by using human IgG myeloma protein and receptor‐specific antibody. M− strains bound IgG and antibody specific for Fc protein, whereas M− strains did not. (ii) Enzyme‐linked immunosorbent assays quantified Fc receptor antigen expression and showed that M+ strains produce more Fc receptor protein than their M− derivatives, (iii) Quantitative RNA dot blots showed that the message for the Fc receptor gene (fcrA) was reduced in M− strains. RNA from M+ strains hybridized to the fcrA probe at a greater dilution than that from their M− counterparts, (iv) Northern hybridization showed that the fcrA transcript is 1200 nucleotides in size and distinct from transcripts for M and SCPA proteins. These data are evidence for the co‐ordinate transcriptional control of the Fc receptor, M protein, and SCPA and show that these proteins co‐ordinately phase‐vary within the same regulatory circuit.
Peptide permeases modulate transformation in Streptococcus pneumoniae
Authors: Pearce B. J. , Naughton A. M. , Masure H. R.
Published: October 2006

To Identify elements participating In the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistentiy transformed 10‐foid less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease‐like protein), which encodes a putative substrate‐binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide‐binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to SpooKA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PIpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]‐palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the pIpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were >90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (ill) the plpA mutation decreases the expression of a competence‐regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate‐permease interaction modulates the process of transformation.