bluuebunny/crossref_metadata_2025 huggingface dataset:
The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin‐like PulG protein into a multiprotein complex
Authors: Pugsley Anthony P. , Possot Odile
Published: October 2006

It has been proposed that the four type IV pilin‐like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans‐periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin‐like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X‐100 + EDTA or at high pH, but not by Triton X‐100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non‐sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross‐linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N‐methylates the PulG precursor, and PulE, a putative ATP‐binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.
Chromosomal domains of supercoiling in Salmonella typhimurium
Authors: Pavitt† Graham D. , Higgins Christopher F.
Published: October 2006

The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains. It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled. We have addressed this question genetically, using a supercoiling‐sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome. We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling. These findings have implications for the organization of the bacterial genome.
Light‐induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region
Authors: McGowan Simon J. , Gorham Hazel C. , Hodgson David A.
Published: October 2006

The carR region encodes a light‐inducible promoter, a negative regulator of the promoter and a trans‐acting activator that controls the light‐inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans‐acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence‐specific DNA‐binding motifs. The sequence of the light‐inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response (‘heat‐shock’) promoters.
Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR
Authors: Sawers Gary
Published: October 2006

Expression of the pfl operon of Escherichia coli Is induced 12‐ to 15‐fold by anaerobiosis and transcription is mediated by seven co‐ordinately regulated promoters. The 5′ non‐translated regulatory region of the operon is approximately 450 bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site‐directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression. Introduction of chromosomal mutations either in the FNR‐binding site or ‐10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the ‐10 region or the putative FNR half‐site located at ‐50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the ‐10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR‐binding site at promoter 6 did reduce transcription from promoter 7. Thus, a cis mutation located 280bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By using pfl–lacZ gene fusions it could be shown that the FNR‐binding site and ‐10 region mutations at promoter 6 abolished FNR‐dependent anaerobic regulation of pfl operon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR‐, but no longer ArcA‐dependent. A construct in which the ‐10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression. This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.
Identification of a gene required for the oxygen‐regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus
Authors: Pollich Michael , Jock Susanne , Klug Gabriele
Published: October 2006

The pigment‐binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild‐type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild‐type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild‐type chromosome or by addition of 10μg I‐1 vitamin B12 to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.
Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression
Authors: Dary A. , Kaiser P. , Bourget N. , Thompson C. J. , Simonet J. M. , Decaris B.
Published: October 2006

Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse‐labelled proteins separated by two‐dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.
Protein–nucleoside contacts in the interaction between the replication terminator protein of Bacillus subtilis and the DNA terminator
Authors: Langley D. B. , Smith M. T. , Lewis P. J. , Wake R. G.
Published: October 2006

The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co‐operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest.
Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at Class II promoters
Authors: West David , Williams Roy , Rhodius Virgil , Bell Andrew , Sharma Naveen , Zou Chao , Fujita Nobuyuki , Ishihama Akira , Busby Stephen
Published: October 2006

The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)‐dependent Class II promoters, where the CRP‐binding site is centred around 411/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP‐dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C‐terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C‐terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.
Secondary sites for integration mediated by the Tn21 integrase
Authors: Francia M. Victoria , Cruz Fernando de la , Lobo Juan M.Garcí
Published: October 2006

The integrase encoded by the integron of the transposon Tn21 can mediate the site‐specific fusion of two plasmids if there is a recombination hot spot (59 bp element) in one of them and the sequence GWTMW in the other. The use of this latter, loosely defined site explains how antibiotic‐resistance genes could first become associated with integrons.
A Mycobacterium leprae‐specific gene encoding an immunologically recognized 45 kDa protein
Authors: De Wit Tobias F. Rinke , Clark‐Curtiss Josephine E. , Abebe Feseha , Kolk Arend H. J. , Janson Anneke A. M. , Agterveld Miranda van , Thole Jelle E. R.
Published: October 2006

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low‐stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T‐cell proliferation assays also demonstrated T‐cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae‐specific immunological and DNA reagents.
Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b
Authors: Kroll J. S. , Langford P. R. , Saah J. R. , Loynds B. M.
Published: October 2006

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen‐metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese‐containing SOD ([Mn]‐SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]‐SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in‐frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]‐SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]‐SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur‐ and Fnr‐binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis‐ responsive activators in H. influenzae gene expression.
Branched Escherichia coli cells
Authors: Åkerlund Thomas , Nordström Kurt , Bernander Rolf
Published: October 2006

Summaryf

We report that the normally rod‐shaped bacterium Escherichia coli can form branched cells. These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g. in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication. Often, chromosome DNA was found to be located in the branch point of the cells. The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased. The genetic background of the strains also affected the tendency to branch. Furthermore, electron microscopy of thin‐sectioned branched cells revealed additional membrane‐like structures, which were not observed in wild‐type cells. Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E. coli are discussed.
Morphogenesis‐independent regulation of actin transcript levels in the pathogenic yeast Candida albicans
Authors: Delbrück Sebastian , Ernst Joachim F.
Published: October 2006

The transcript level of the Candida albicans ACT1 gene (encoding actin) is strongly regulated during induction of hyphal morphogenesis. ACT1 mRNA declines rapidly during starvation pretreatment and quickly recovers in media inducing morphogenesis. The C. albicans URA3 and LEU2 mRNAs, as well as an ACT1 promoterILAC4 fusion, are regulated similarly. The regulation of ACT1ILAC4 and unaltered mRNA stabilities suggest transcriptional regulation during morphogenesis. However, by individually testing morphogenesis induction parameters, it is shown that starvation and growth phase, but not hyphal formation, are responsible for ACT1 transcript regulation; this conclusion is confirmed by analyses of morphological mutants and by inhibition of hyphal development. Thus, the specific morphogenesis‐induction conditions, but not morphogenesis per se, affect transcript levels in C. albicans.
Transaldolase mutants in the yeast Kluyveromyces lactis provide evidence that glucose can be metabolized through the pentose phosphate pathway
Authors: Jacoby J. , Hollenberg C. P. , Heinisch J. J.
Published: October 2006

We have isolated the gene encoding transaldolase from Kluyveromyces lactis (KITALI) by screening a genomic library of this yeast using the TAL1 gene of Saccharomyces cerevisiae as a radioactive probe. The clone isolated contained an open reading frame of 1002 bp, encoding a protein with 76% identical residues in the deduced amino acid sequences as compared to Tal from S. cerevisiae. KITAL1 can complement a tal1 deletion of S. cerevisiae for enzymatic activity. The transcription start of KITAL1 was located at ‐69 bp relative to the ATG translation start codon. Deleting a large part of the open reading frame from the genome did not lead to any obvious phenotype. Transaldolase was not produced in such mutants as shown by immunological detection. In combination with a double null‐mutant in the genes encoding the phosphofructokinase subunits in K. lactis (Klpfk1 Klpfk2 Kltal1), the cells lost their ability to grow on glucose. We take this as strong evidence that glucose is metabolized via the pentose phosphate pathway in this yeast when glycolysis is blocked. In addition, by tetrad analysis we detected a close linkage to KIPFK1 and inferred that KITAL1 is localized on chromosome I.
Interaction of a redox‐sensitive DNA‐binding factor with the 5′‐flanking region of the micF gene in Escherichia coli
Authors: Gidrol Xavier , Farr Spencer
Published: October 2006

The product of the micF gene is an endogenous anti‐sense RNA which down‐regulates the expression of a major outer membrane protein, OmpF, in E. coli. We report here that two DNA‐binding factors compete for the same site in the promoter region of the micF gene: RSBF, a high‐affinity redox‐sensitive DNA‐binding factor that responds to an active oxygen species other than hydrogen peroxide or superoxide anions; and HRBF a heat‐resistant DNA‐binding factor. Both RSBF and HRBF bind to the same DNA sequence, 5′‐TTAAAATCAATAACTTATTCTTAA3‐′, located upstream of the transcription start site of the micF gene. We present evidence that RSBF could be the controlling factor of a novel regulon involved in the response to oxidative stress in E. coli.
Repair of cyclobutane pyrimidine dimers and 6‐4 photoproducts in the fission yeast Schizosaccharomyces pombe
Authors: McCready Shirley , Carr Antony M. , Lehmann Alan R.
Published: October 2006

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6‐4 photoproducts) in wild‐type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision‐defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision‐defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild‐type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.
The ompS gene of Vibrio cholerae encodes a growth‐phase‐dependent maltoporin
Authors: Lå Hannu , Palva E. Tapio
Published: October 2006

The outer membrane of Vibrio cholerae contains a maltose‐inducible major protein, OmpS (43 kDa), that is common to different isolates. Nucleotide sequence analysis of the corresponding structural gene, ompS, revealed an open reading frame encoding a 412‐amino‐acid polypeptide. The amino acid sequence of OmpS is similar to that of LamB, the Escherichia coli maltoporin, and to ScrY of Klebsilla pneumoniae, although the antigenic determinants of these proteins are different. The cloned ompS gene complemented an ompS mutation of V. cholerae and the corresponding polypeptide could function as a maltoporin in a LamB‐ mutant of E. coli. The promoter region of ompS is highly homologous to the malK‐lamB promoter of E. coli and the ompS gene is controlled by MalT in E. coli. This indicates that the same kind of regulatory mechanism is used to activate the ompS expression in V. cholerae and malK‐lamB expression in E. coli. An ompS‐lacZ transcriptional fusion was used to demonstrate a dual control in ompS expression; the ompS gene is responsive to the inducers maltose and trehalose but in their absence it is also expressed in response to growth‐phase. These different modes of induction might be of importance during different stages of V. cholerae infection.
Linear plasmids and chromosomes in bacteria
Authors: Hinnebusch Joe , Tilly Kit
Published: October 2006

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram‐positive bacteria, and Gram‐negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram‐positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.
The chromosomal DNA of Streptomyces lividans 66 is linear
Authors: Lin Yi‐Shing , Kieser Helen M. , Hopwood David A. , Chen Carton W.
Published: October 2006

Two copies of a DNA sequence similar or identical to one end of the linear plasmid SLP2 were found on the Streptomyces lividans chromosome. Restriction mapping showed that these sequences represented free ends. Electrophoretic retardation and glass‐binding studies indicated that the telomeres carry covalently bound proteins. Moreover, the chromosome migrated as an 8Mb linear DNA in pulsed‐field gel electrophoresis. A similar finding with the chromosomes of six other Streptomyces species suggested that a linear chromosome may be characteristic of the genus. The S. lividans chromosome can be circularized by joining the two ends by artificial targeted recombination or by spontaneous deletions spanning both telomeres. Thus the chromosome appears to be able to exist, in viable bacteria, as a linear or a circular molecule.
The genus‐specific lipopolysaccharide epitope of Chlamydia is assembled in C. psittaci and C. trachomatis by glycosyltransferases of low homology
Authors: Mamat U. , Baumann M. , Schmidt G. , Brade H.
Published: October 2006

Chlamydiae possess a genus‐specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3‐deoxy‐d‐manno‐octulosonic acid (Kdo) trisaccharide of the sequence αKdo‐(2→8)–αKdo–(2→4)‐αKdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi‐functional Kdo‐transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two α2→4‐linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in a α2→8‐linkage to the parental LPS, as determined by SDS–PAGE and Western blot analysis using a monoclonal antibody against the genus‐specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity.
The conserved proline‐rich Motif is not essential for energy transduction by Escherichia coliTonB protein
Authors: Larsen Ray A. , Wood Gwendolyn E. , Postle Kathleen
Published: October 2006

TonB protein functions as an energy transducer, coupling cytoplasmic membrane electrochemical potential to the active transport of vitamin B12 and Fe(III)–siderophore complexes across the outer membrane of Escherichia coli and other Gram‐negative bacteria. Accumulated evidence indicates that TonB is anchored in the cytoplasm, but spans the periplasmic space to interact physically with outer membrane receptors. It has been presumed that this ability is caused by a conserved (Glu–Pro)n–(Lys–Pro)m repeat motif, predicted to assume a rigid, linear conformation of sufficient length to reach the outer membrane. Based on in vitro studies with synthetic peptides and purified FhuA outer membrane receptor, it has been suggested that this region contains a site that directly binds outer membrane receptors and is essential for energy transduction. We have found a TonB lacking the (Glu–Pro)n–(Lys–Pro)m, repeat motif (TonBΔ(66–100)). TonBΔ(66–100) is fully capable of irreversible 80 adsorption, except under physiological circumstances where the periplasmic space is expanded. Based on the ability of TonBΔ(66–100) to interact with outer membrane receptors and components of the energy transduction apparatus under normal physiological conditions, it is evident that the TonB proline‐rich region has no role in energy transduction other than to provide a physical extension sufficient to reach the outer membrane.
Differential regulation of two myo‐inositol transporter genes of Saccharomyces cerevisiae
Authors: Nikawa J. , Hosaka K. , Yamashita S.
Published: October 2006

We previously isolated two distinct Saccharomyces cerevisiae myo‐inositol transporter genes, ITR1 and ITR2 (Nikawa et al., 1991). Here, we studied the regulation of their expression by measuring steady‐state mRNA levels and β‐galactosidase activities of lacZ fusion genes under different conditions. The results show that the expression of the two ITR genes is differently regulated: ITR1 was repressed by inositol and choline whereas ITR2 was constitutive. Deletion analysis of the ITR1 upstream region and comparison with the upstream regions of other genes involved in phospholipid synthesis indicate that the octamer sequence 5′‐TTCACATG‐3′ is important for the expression and inositol/choline regulation of the ITR1 gene.
The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis
Authors: Sommer Suzanne , Bailone Adriana , Devoret Raymond
Published: October 2006

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA‐mediated recombination. Homologous recombination in an Hfr x F‐ cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.‐damaged F‐lac with the lac gene of an F‐ recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.
DNA loop formation between Nag repressor molecules bound to its two operator sites is necessary for repression of the nag regulon of Escherichia coli in vivo
Authors: Plumbridge Jacqueline , Kolb Annie
Published: October 2006

Binding sites for the Nag repressor overlap the transcription start sites of the divergent nagE and nagB genes, such that the centres of the sites are separated by nine turns of the B‐DNA helix. Mutations which prevent repressor binding to either site or alter the phasing of the binding sites result in simultaneous derepression of both genes. An additional mutation which restores the phasing of the two sites permits repression. These observations show that repression is the result of co‐operative binding of the repressor to its two sites, resulting in the formation of a loop of DNA.
Hydrophobic domains affect the collagen‐binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica
Authors: Tamm Anu , Tarkkanen Ann‐Mari , Korhonen Timo K. , Kuusela Pentti , Toivanen Paavo , Skurnik Mikael
Published: October 2006

The YadA surface protein of enteropathogenic Yersinia species contains two highly hydrophobic regions: one close to the amino terminal, and the other at the carboxy‐terminal end of the YadA polypeptide. To study the role of these hydrophobic regions, we constructed 66 bp deletion mutants of the yadA genes of Yersinia enterocolitica serotype O:3 strain 6471/76 (YeO3) and of O:8 strain 8081 (YeO8). The mutant proteins, YadAYeO3‐Δ83–104 and YadAYeO8‐Δ80–101, lacked 22 amino acids from the amino‐terminal hydrophobic region, formed fibrillae and were expressed on the cell surface. Bacteria expressing the mutated protein lost their auto‐agglutination potential as well as their collagen‐binding property. Binding to fibronectin and laminin was affected differently in the YeO3 and the YeO8 constructs. The deletion did not influence YadA‐mediated complement inhibition. Loss of the collagen‐binding property was associated with loss of virulence in mice. We also constructed a number of YadAYeO3 deletion mutants lacking the hydrophobic carboxy‐terminal end of the protein. Deletions ranging from 19 to 79 amino acids from the carboxy terminus affected polymerization of the YadA subunits, and also resulted in the loss of the YadA expression on the cell surface. This suggests that the carboxy terminus of YadA is involved in transport of the protein to the bacterial outer surface.
Pilus‐facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin
Authors: Virji Mumtaz , Saunders Jon R. , Sims Gail , Makepeace Katherine , Maskell Duncan , Ferguson David J. P.
Published: October 2006

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non‐piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper‐adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS‐PAGE. Hyper‐adherent pilins shared unique changes in sequence including substitution of Asn‐113 for Asp‐113 and changes from Asn‐Asp‐Thr‐Asp to Thr‐Asp‐Ala‐Lys at residues 127‐130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N‐glycosylation motif Asn‐X‐Ser/Thr and could potentially be glycosylated post‐translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS‐PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn‐60‐Asn‐61‐Thr‐62 in an adherent variant was replaced with Asp‐60‐Asn‐61‐Ser‐62 in a non‐adherent variant. Fully adherent backswitchers obtained from the non‐adherent variant always regained Asn‐60 but retained Ser‐62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N‐linked glycosylation sites on the pilin subunit.
Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli
Authors: Delgado Jorge , Forst Steven , Harlocker Susan , Inouye Masayori
Published: October 2006

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp‐11, Asp‐12 and/or Asp‐55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino‐terminal domain of OmpR with [3H]‐NaBH4 indicated that Asp‐55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp‐55 is the primary phosphate acceptor site in OmpR, Asp‐11 may also serve as a phosphorylation site, particularly in the absence of Asp‐55.
Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method
Authors: Groenen Peter M. A. , Bunschoten Annelies E. , Soolingen Dick van , Errtbden Jan D. A. van
Published: October 2006

Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR‐PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR‐PCR allows the detection of M. tuberculosis and strain differentiation at the same time.
Viability and preliminary in vivo characterization of site directed mutants of Escherichia coli single‐stranded DNA‐binding protein
Authors: Carlini Leslie E. , Porter Ronald D. , Curth Ute , Urbanke Claus
Published: October 2006

Site‐directed mutations involving selected amino acids of Escherichia coli single‐stranded DNA‐binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb* control, and both the ssbW54S‐ and ssbH55K‐containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb‐1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.
Sensory rhodopsin‐controlled release of the switch factor fumarate in Halobacterium salinarium
Authors: Montrone Marco , Marwan Wolfgang , Grünberg Heidi , Mußeleck Sabine , Starostzik Christine , Oesterhelt Dieter
Published: October 2006

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low‐molecular‐mass fraction (<5 kDa) of the lysate was assayed enzymatically and a light‐induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light‐induced release was not detected in a mutant defective in sensory rhodopsin‐l and ‐II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin‐II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light‐induced fumarate release is at least 10‐times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.
A gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae
Authors: Larsson Katrin , Ansell Ricky , Eriksson Peter , Adler Lennart
Published: October 2006

Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive gtycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1‐osg4). The osg1‐1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD+‐dependent sn‐glycerol 3‐phosphate dehydrogenase (GPD), an enzyme in the glycerol‐producing pathway. Complementation of the osg1‐1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47‐50% identity with GPD from other sources. Micro‐sequencing of the N‐terminus of purified S. cerevisiae GPD revealed a 20‐amino‐acid sequence that was identical to a nucleotide‐deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N‐terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre‐sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.
Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22
Authors: Frère Jacques , Novel Madeleine , Novel Georges
Published: October 2006

pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non‐transcribed region, the replication origin, followed by a gene encoding a putative 388‐amino‐acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co‐resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co‐existence of numerous related replicons in lactococcal strains.
Hyper‐invasive mutants define a novel Pho‐regulated invasion pathway in Escherichia coli
Authors: Sinai A. P. , Bavoil P. M.
Published: October 2006

We have isolated two transposon insertion mutations of the pst–phoU operon which result in the constitutive expression of the phoA gene product, alkaline phosphatase. The two mutations also render Escherichia coli invasive towards cultured HEp‐2 cells and define a novel Pho‐regulated invasion pathway. The presence of the large‘invasion’plasmid derived from an entero‐invasive E. coli (EIEC) clinical isolate in these mutants leads to enhanced invasiveness toward cultured HEp‐2 cells, a phenomenon referred to as the‘hyper‐invasive’phenotype. Transduction of a pst–phoU insertion mutation into clinical isolates of EIEC and Shigella flexneri results in constitutive PhoA expression and coupled hyper‐invasiveness in the former but not the latter. We speculate that the Pho‐regulated invasion pathway described here, while silent in bacteria grown in standard laboratory rich media, may become functional in the host when invasive bacteria encounter nutrient starvation and/or other related stress conditions.
The Cro‐like Apl repressor of coliphage 186 is required for prophage excision and binds near the phage attachment site
Authors: Dodd Ian B. , Reed Michael R. , Egan J. Barry
Published: October 2006

The Apl protein of the temperate coliphage 186 represses transcription of the immunity repressor gene and down‐regulates lytic transcription. It is shown here that an apl‐ mutant is competent for lytic development and establishes lysogeny normally but is defective in excision of the prophage. The Apl protein binds between the lytic and lysogenic promoters and also near the phage attachment site, suggesting that its role in excision is direct. Apl thus appears to act as an excisionase as well as a repressor. The pattern of Apl‐induced DNase I enhancements indicates that the DNA is bent by Apl. Potential Apl recognition sequences are identified; these sequences are directly repeated several times across each binding region and are spaced 10 or 11 bases apart, suggesting that Apl binds to one face of the DNA helix.
Copper uptake and resistance in bacteria
Authors: Cooksey Donald A.
Published: October 2006

Copper ions are essential for bacteria but can cause a number of toxic cellular effects if levels of free ions are not controlled. Investigations of copper‐resistant bacteria have revealed several mechanisms, mostly plasmid‐determined, that prevent cellular uptake of high levels of free copper ions. However, these studies have also revealed that bacteria apparently have efficient chromosomally encoded systems for uptake and management of trace levels of copper. This review will explore the relationship of copper uptake systems to resistance mechanisms and the possibility that copper resistance has evolved directly through modification of chromosomal copper uptake genes.
Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila
Authors: Berger Karen H. , Isberg Ralph R.
Published: October 2006

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage‐like cultured cells. One class of mutants was defective for both inhibition of phagosome–lysosome fusion and association of host cell organelles with bacteria‐containing phagosomes (‘recruitment’). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild‐type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.
Membrane topology of MaIG, an inner membrane protein from the maltose transport system of Escherichia coli
Authors: Dassa Elie , Muir Susie
Published: October 2006

In Escherichia coli, the binding protein‐dependent transport system for maltose and maltodextrins is composed of five proteins — LamB, MaIE, MaIF, MaIG and MaIK — located in the three layers of the bacterial envelope. Proteins MaIF and MaIG are hydrophobic inner membrane components mediating the energy‐dependent translocation of substrates into the cytoplasm. In this paper, we analyse the topology of the MaIG protein by using methods based on the properties of fusions between maIG and‘phoA, a truncated gene encoding alkaline phosphatase lacking its translation initiation and exportation signals. Fusions were obtained by using either phage λTnphoA or by constructing in vitro fusions located randomly within the maIG gene. The deduced topological model suggests that MaIG spans the membrane six times and has its amino‐ and carboxy‐termini in the cytoplasm. These results will be helpful for the interpretation of the phenotypes of mutants in maIG.