bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA‐binding domain
Authors: Brown David P. , Ganova‐Raeva Lilia , Green Brian D. , Wilkinson Shane R. , Young Michael , Youngman Philip 
Published: October 2006

SummarySpo0A is a phosphorylation‐activated transcription factor of Bacillus subtilis. It is a member of the response regulator super‐family of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of speculation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix‐turn‐helix motif, and, therefore, represents the actual DNA‐contacting surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene‐disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spolVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation‐specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A‐like gene in non‐endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A‐like proteins may be exclusive to the endospore‐forming bacteria.
The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation
Authors: Biesen Tim van , Frost Laura S. 
Published: October 2006

SummaryMost of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F‐like conjugative plasmids. Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system. The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression. Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target. Using electrophoretic mobility shift assays, we have shown that FinO is an RNA‐binding protein that binds to one of the two stem‐loops in FinP and to its complementary structure in traJ mRNA. This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP‐ traJ mRNA duplex fivefold. Thus, TraJ expression appears to be influenced by a unique RNA‐protein interaction that precedes duplex formation between the FinP anti‐sense RNA and its target traJ mRNA.
Multicopy single‐stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli
Authors: Maas Werner K. , Wang Chi , Lima Tania , Zubay Geoffrey , Lim Dongbin 
Published: October 2006

SummaryRetrons are genetic elements that encode multicopy single‐stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single‐stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem‐loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem‐loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.
High‐frequency S‐layer protein variation in Campylobacter fetus revealed by sapA mutagenesis
Authors: Blaser Martin J. , Wang Enze , Tummuru Murali K. R. , Washburn Ronald , Fujimoto Shuji , Labigne Agnès 
Published: October 2006

Summary Campylobacter fetus utilizes paracrystalline surface (S‐) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full‐length S‐layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full‐length S‐layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10‐2. Immunoblots of survivors showed that phenotypic reversion involving high‐level production of full‐length (98, 127 or 149 kDa) S‐layer proteins had occurred. Revertants were serum resistant but caused approximately 10‐fold less bacteraemia in orally challenged mice than did the wild‐type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high‐frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.
The pAMβ1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene
Authors: Chatelier E. Le , Ehrtich S. D. , Jannière L. 
Published: October 2006

SummarypAMβ1 is a low‐copy‐number, promiscuous plasmid from Gram‐positive bacteria that replicates by a unidirectional theta‐type mode. Its replication is initiated by an original mechanism, involving the positive rate‐limiting RepE protein. Here we show that the pAMβ1‐encoded CopF protein is involved in negative regulation of the plasmid copy number. CopF represses ‐10‐fold the transcription initiated at the promoter of the repE gene and binds to a 31 bp segment which is located immediately upstream of the ‐35 box of the repE promoter. We propose that CopF inhibits initiation of transcription at the repE promoter by binding to its operator.
The copR gene product of plasmid p1P501 acts as a transcriptional repressor at the essential repR promoter
Authors: Brantl Sabine 
Published: October 2006

SummaryThe amount of the rate‐limiting replication initiator protein RepR of plasmid p1P501 is negatively controlled by an antisense RNA (RNAIII) and a dispensable protein (CopR). Deletions or mutations in either component cause a 10‐20‐fold copy number increase. RNAIII induces transcription attenuation of the repR mRNA: the mode of CopR action remained unclear. To test the function of CopR, transcriptional fusions of promoters pI, pII and pIII with lacZ were integrated into the Bacillus subtilis chromosome. CopR and/or RepR were supplied in trans, and LacZ synthesis measured. The results show that CopR represses the repR promoter pII. Neither CopR nor RepR autoregulate their promoters. Gel mobility shift assays indicate that CopR binds to a 44 bp DNA fragment comprising the inverted repeat upstream of pII.
Early stages in development of the Escherichia coli cell‐division site
Authors: Cook William R. , Rothfield Lawrence I. 
Published: October 2006

SummaryDevelopment of the Escherichia coli cell division site was studied in wild‐type cells and in non‐septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non‐septate filaments of FtsZ‐ cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild‐type cells and FtsZ‐ filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.
Development of the cell‐division site in FtsA‐filaments
Authors: Cook William R. , Rothfield Lawrence I. 
Published: October 2006

SummaryEarly changes at cell‐division sites were studied in non‐septate filaments induced by growth of ftsATs mutant cells under non‐permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division‐site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.
Identification of an ATP‐binding cassette transport system required for translocation of lipopolysaccharide O‐antigen side‐chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1
Authors: Bronner Dorothea , Clarke Bradley R. , Whitfield Chris 
Published: October 2006

SummaryThe rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D‐galactan I component of the lipopolysaccharide O‐antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP‐binding domain and shares homology with several proteins which function as ATP‐binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane‐spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K‐1 and K‐5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two‐component ABC (ATP‐binding cassette) transporters. E. coli K‐12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O‐antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O‐polysaccharide transport is restored and smooth D‐galactan l‐substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D‐galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane‐spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.
Mode of action of LciA, the lactococcin A immunity protein
Authors: Venema K. , Haverkort R. E. , Abee T. , Haandrikman A. J. , Leenhouts K. J. , de Leij L. , Venema G. , Kok J. 
Published: October 2006

SummaryMonoclonal antibodies were raised against a fusion between the Escherichia coli maltose‐binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane‐associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A.The monoclonal antibody specifically reacted with right‐side‐out vesicles obtained from a strain producing the immunity protein. It did not react with inside‐out vesicles of the same strain, or with right‐side‐out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right‐side‐out vesicles obtained from a strain producing only LciA.The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right‐side‐out vesicles by proteinase K, it is located at the outside of the cell.The immunity protein contains a putative a‐amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C‐terminal part of the protein, containing the epitope, is on the outside of the cell.Vesicle‐fusion studies together with leucine‐uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.
High‐frequency homologous plasmid‐plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces
Authors: Xiao J. , Melton R. E. , Kieser T. 
Published: October 2006

SummaryNon‐transmissible derivatives of the Streptomyces multi‐copy plasmid pIJ101 were mobilized, by co‐integrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low‐copy‐number Streptomyces coeli‐color A3(2) plasmid SCP2*. Efficient co‐integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co‐integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.
Identification of IS 1206, a Corynebacterium glutamicum IS3‐related insertion sequence and phylogenetic analysis
Authors: Bonamy Celine , Labarre Jean , Reyes Oscar , Leblon Gèrard 
Published: October 2006

SummaryIntegration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3‐related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.
Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus
Authors: Collazo Carmen M. , Zierler Michael K. , Gatan Jorge E. 
Published: October 2006

SummaryWe have carried out a functional analysis of invl and invJ, two Saimonella typhimurium genes required for this organism to gain access So cultured mammalian cells. These genes are located immediately downstream of invC, a previously identified gene also required for bacterial invasion. Non‐polar mutations in either of these genes rendered S. typhimurium severely defective for entry into cultured epithelial cells, although these mutations did not affect the ability of these organisms to attach to those cells. Nucleotide sequence analysis revealed that the invl and invJ genes encode proteins with molecular weights of 18077 and 36415, respectively. Polypeptides of similar sizes were observed when these genes were expressed in a bacteriophage T7 RNA polymerase‐based expression system. Comparison of the predicted sequences of Invl and InvJ with translated sequences in the existing databases indicated that these proteins are Identical to the previously identified S. typhimurium SpaM and SpaN proteins. Further analysis of these sequences revealed regions of homology between Invl and the N‐terminus of lpaB of Shigella spp. and between InvJ and EaeB of enteropathogenic Escherichia coli. Localization studies by immunoblot analysis indicated that InvJ is secreted to the culture supernatant, a surprising finding since this protein also lacks a typical signal sequence. Mutations in invG and inyC, two members of the Salmonella inv locus, effectively prevented the transport of InvJ to the culture supernatant. Thus, InvJ is the first identified target of the protein secretion apparatus encoded in the inv locus and therefore a candidate to have effector functions related to bacterial entry.
A small gene, designated comS, located within the coding region of the fourth amino acid‐activation domain of srfA, is required for competence development in Bacillus subtilis
Authors: Hamoen Leendert W. , Eshuis Hans , Jongbloed Jan , Venema Gerard , van Sinderen Douwe 
Published: October 2006

SummaryThe valine‐activation domain‐encoding portion of the srfA locus (srfA‐d4) is not only involved in the non‐ribosomal synthesis of surfactin, but is also required for the regulation of competence development. In this study we show that impairment of the adenylation activity of the valine‐activating domain did not affect competence development. Deletion analysis and complementation studies delineated the competence‐required portion of srfA‐d4 to a 168bp fragment, which contains a small open reading frame (ORF), designated comS, encoding a polypeptide of 46 amino acids, embedded within, but translated in, a frame different from that of srfA‐d4. Introduction of an amber mutation in the comS‐coding frame prevented competence development, demonstrating the involvement of comS in this prokaryotic specialization process.
Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial ceils
Authors: Geme Joseph W. St. , Cutter David 
Published: October 2006

Summary Hemophilus influenzae type b is a Gram‐negative bacillus that initiates infection by colonizing the upper respiratory tract. Previous studies have established that H. influenzae haemagglutinating pili possess adhesive properties and influence the process of colonization. Additional studies suggest the presence of a second H. influenzae adhesin distinct from haemagglulinating pili. In the present study, we examined a non‐piliated H. influenzae type b strain by transmission electron microscopy and visualized occasional short, thin, surface fibrils. Subsequently, we isolated a spontaneous mutant that lacked surface fibrils and was deficient in adherence to cultured human epithelial cells. Using a cloning strategy that exploited this mutant, we isolated a fragment of DNA that promotes in vitro adherence to human epithelial cells when expressed in laboratory strains of Escherichia coli. Mutagenesis of this fragment in a series of H. influenzae type b strains resulted in loss of expression of surface fibrils and a marked decrease in attachment. Furthermore, restoration of surface fibril expression was associated with reacquisition of wild‐type levels of adherence. Our results are consistent with the conclusion that H. influenzae type b surface fibrils have adhesive capacity. We speculate that these organelles facilitate colonization of the human respiratory tract.
A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1 is essential for virulence: histamine is a precursor in the biosynthesis of anguibactin
Authors: Tolmasky Marcelo E. , Actis Luis A. , Crosa Jorge H. 
Published: October 2006

SummaryWe have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJMl which encodes a histidine decarboxlase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44 259.69 Da. The amino acid sequence has extensive homology with the pyridoxal‐P‐dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3‐HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJMI Iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron‐limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore. immunoblot analysis of production of β‐galactosidase by V. anguillarum strains carrying the lacZ fusions demonstrated that expression of histidine decarboxylase is not regulated by the iron concentration of the medium.