bluuebunny/crossref_metadata_2025_split huggingface dataset:
Beta‐lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus
Authors: Reeves Philip J. , Douglas Paul , Salmond George P. C. 
Published: October 2006

SummaryThe out gene cluster of Erwinia spp. encodes the proteins of the general secretory pathway (GSP) apparatus that is required for pectinase and cellulase secretion. We have used fusions between Erwinia carotovora subsp. carotovora (Ecc) out genes and the topology probe blaM to assess the ability of Out protein regions to export BlaM across the cytoplasmic membrane in Escherichia coli and Ecc. For the outO gene product (an NMePhe peptidase), seven transmembrane regions have been identified and one more is predicted. The region of OutO with the highest level of hydrophilicity is likely to exist as a large cytoplasmic loop, located between two hydrophobic domains, and is positioned towards the N‐terminus of the protein. When BlaM was fused on the C‐terminal side of the last hydrophobic stretch of OutO, the resulting hybrid protein transferred the BlaM moiety to the periplasm whilst retaining OutO activity. Removal of a portion of this hydrophobic stretch resulted in the loss of OutO activity, suggesting that there are tight constraints on the topological integrity of OutO for maintaining catalytic function. When outG, ‐H, ‐I, ‐J, ‐K and ‐N were fused to blaM, the resulting phenotype suggested that the majority of each protein was targeted to the periplasm. Our results indicate that these six Out proteins, when produced by E. coli or Ecc, each adopt, at least temporarily, a type II bitopic conformation in the cytoplasmic membrane. For OutG, ‐H, ‐I and ‐J this probably represents the membrane topology prior to processing by OutO in Ecc. When produced in vivo from a T7 gene 10 promoter construct, the outG product was processed in Ecc whereas the outO mutant RJP249 failed to process pre‐OutG. BlaM fusions positioned on the C‐termtnal side of the hydrophobic stretches of pre‐OutG, ‐H, ‐I, and ‐J were processed by wild‐type Ecc but not RJP249 or E. coli DHL Thus the periplasmic domains of these proteins play no role in the peptidase cleavage reaction. An OutG‐BlaM fusion construct was used to demonstrate NMePhe peptidase activity in other bacterial strains including E. carotovora subsp. carotovora (ATCC39048), E. carotovora subsp. atroseptica (SCR11043) and Erwinia chrysanthemi (3937).
Replication of a Bacillus subtilis oriC plasmid in vitro
Authors: Moriya S. , Firshein W. , Yoshikawa H. , Ogasawara N. 
Published: October 2006

SummaryWe constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis‐elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi‐conservative and only one round of replication occurred within 15min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2‐dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and unidirectional replication occurred in vitro.
MycDB: an integrated mycobacterial database
Authors: Bergh Staffan , Cole Stewart T. 
Published: October 2006

SummaryAs part of ongoing efforts to Investigate the molecular biology of the human pathogens in the genus Mycobacterium, a customized database was developed specifically for these organisms and implemented in ACEDB database manager software. The data loaded include the IMMYC Antigen List, details of reagents available from the CDC/WHO Antibody Bank, more than 1 Mb of sequences of mycobacterial genes and proteins from public databases, the physical maps of Mycobacterium leprae and Mycobacterium tuberculosis developed at the institut Pasteur, as well as a subset of the references found in MedLine. The ACEDB software allows both quick and intuitive access to the data and to connections between facts by a simple mouse‐driven interface, as well as by more powerful query mechanisms.
The gene of the N‐acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis
Authors: Margot Philippe , Mauël Catherine , Karamata Dimitri 
Published: October 2006

Summary lytD, the structural gene of the Bacillus subtilis 168 N‐acetylglucosaminidase was localized at 310°, next to the tagABC operon. Sequence analysis revealed a monocistronic operon encoding a 95.6 kDa protein endowed with an export signal, the cleavage of which yields the monomer polypeptide (92.8 kDa) of the dimeric active form of the enzyme. Transcription is initiated at a sigma‐D (σD)‐dependent promoter and ends at a terminator common to lytD and the divergently transcribed tagABC operon. In addition, we report the sequence of the adjacent upstream ORF, transcribed in the same direction as lytD, which shows significant homology to phosphomannose isomerase‐encoding genes. Cell separation, motility, autolysis, cell wall turnover and growth were not affected in strains devoid of the N‐acetylglucosaminidase. A mutant deficient in the two most abundant autolysins, i.e. the LytC amidase and the glucosaminidase, exhibited the phenotype of the amidase‐deficient strains, revealing their non‐requirement for growth. This conclusion raises two fundamental questions: how does the cell undo the highly cross‐linked peptidoglycan so as to be able to grow, and what is the rote of the considerable amount of autolysin normally present? Possible answers to these questions are discussed.
Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis
Authors: Dybvig Kevin , Yu Hullan 
Published: October 2006

SummaryAn invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R‐M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R‐M enzymes previously found only in enteric bacteria.
Nark is a nitrite‐extrusion system involved in anaerobic nitrate respiration by Escherichia coli
Authors: Rowe John J. , Ubbink‐Kok Trees , Molenaar Douwe , Konings Wil N. , Driessen Arnold J. M. 
Published: October 2006

Summary Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest‐lived radioactive isotope of nitrogen, 13N‐nitrate (half‐life = 9.96 min) and the nitrite‐sensitive fluorophore N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.
Molecular basis of the optochin‐sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+‐ATPases
Authors: Fenoll Asunción , Muñoz Rosario , Garcia Ernesto , de la Campa Adela G. 
Published: October 2006

SummaryThe gene responsible for the optochin‐sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H+‐ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin‐sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by inter‐species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane‐bound ATPase activity of the S. pneumoniae wild‐type (OptS) strains, and found a 100‐fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.
Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba
Authors: Cázares Febe , Manning‐Cela Rebecca , Meza Isaura 
Published: October 2006

SummaryRibosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment‐length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non‐transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non‐pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA‐containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.
Control of the LexA regulon by pH: evidence for a reversible inactivation of the LexA repressor during the growth cycle of Escherichia coli
Authors: Dri Anna‐Maria , Moreau Patrice L. 
Published: October 2006

SummaryThe LexA repressor controls the expression of several genes, including lexA, recA, and sfiA, which are induced when exponentially growing bacteria are exposed to DNA‐damaging agents, Induction of this so‐called SOS response takes place while LexA is cleaved in a reaction that requires the RecA protein and damaged DNA. We have shown that large fluctuations in the cellular concentration of the LexA repressor and in the rate of transcription of the sfiA gene also occur spontaneously during bacterial growth in complex medium such as LB. The possibility that changes in external or internal pH may explain these fluctuations has been explored. A consistent pattern was established whereby conditions leading to either increased or decreased pH were associated with altered expression of the LexA and SfiA genes. These data can be explained by a model in which the LexA repressor exists in either of two forms in equilibrium: a form favoured at homeostatic internal pH, which has a low affinity for the operators of LexA‐controlled genes; and a form accumulated in response to a transient decrease in internal pH, which has a high affinity for operators.
Novel growth rate control of dam gene expression in Escherichia coli
Authors: Rasmussen Lene Juel , Marinus M. G. , Lobner‐Olesen Anders 
Published: October 2006

SummaryTranscription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single‐copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical σ70 promoter with 18bp spacing between the ‐10 and ‐35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta‐galactosidase specific activity from a single‐copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde‐4), obtained by miniTn 10 insertion, showed the same level of β‐galactosidase activity at all growth rates tested, in contrast, growth rate‐dependent expression of the rrnB gene was unaffected by cde‐4 confirming the different modes of regulation. The cde‐4::mint Tn 10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.
The regulatory RNA gene micF is present in several species of Gram‐negative bacteria and is phylogenetically conserved
Authors: Esterling Lisa , Delihas Nicholas 
Published: October 2006

Summary micF RNA post‐transcriptionally regulates Escherichia coli outer membrane protein F (OmpF), in response to temperature increase and other environmental stress conditions, by binding to ompF mRNA and destabilizing the message. Southern analyses show that the micF gene is present in related Gram‐negative bacteria, including Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, Northern analyses Indicate that micF RNA and ompF mRNA levels are thermally regulated in several related species in a manner similar to the thermoregulation in Escherichia coli DNA sequences from Salmonella typhi, Salmonella typhimurium, and Klebsiella pneumoniae show greater than 96% homology in the micF gene when compared to the Escherichia coli micF sequence. Upstream of micF, sequences show considerable variation, although several distinct regions are highly conserved. Some of these conserved regions correspond to known binding sites for the transcription factor OmpR and the DNA‐binding protein integration host factor. In addition, E. coli micF RNA incubated with protein extracts from other species forms hetero‐logous ribonucleoproteins (RNPs). The formation of these heterologous RNPs indicates both the presence of micF RNA‐binding protein homologues in other species and a conservation of RNA‐protein recognition sites. This work demonstrates that the micF RNA regulatory system is present in other Gram‐negative bacterial species and that this system appears to be phylogenetically conserved.
Isolation and characterization of a carboxysome shell gene from Thiobacillus neapolitanus
Authors: English Robert S. , Lorbach Stanley C. , Qin Xiang , Shively Jessup M. 
Published: October 2006

SummaryThe gene coding for the major carboxysome shell peptide (csoS1) from Thiobacillus neapolitanus has been isolated and sequenced. Oligonucleotide primers for polymerase chain reaction (PCR) amplification of the 5’end of the gene were made possible by amino acid sequencing of the N‐terminal residues of the shell peptide. A 41 bp PCR product was used as a probe to isolate the gene. The deduced amino acid composition of the 216 bp gene shows a high degree of hydrophobicity. The gene is located within a series of three repeated regions of DNA and appears to have arisen via gene duplication. The transcript of csoS1 is approximately 400 bases in length. The shell peptide shares significant homology with Synechococcus open reading frames implicated in carboxysome structure/assembly. These open reading frames and csoS1 are related and are probably members of a carboxysome gene family.
The Lactococcus lactis sex‐factor aggregation gene cluA
Authors: Godon Jean‐Jacques , Jury Karen , Shearman Claire A. , Gasson Michael J. 
Published: October 2006

SummaryA gene, cluA, was cloned from the chromosomally located sex factor of Lactococcus lactis MG1363. Sequence analysis revealed significant homology with previously described aggregation proteins in Enterococcus and Streptococcus species. The possibility that cluA was an equivalent protein involved in cell aggregation between donor and recipient bacteria during lactococcal conjugation was confirmed by its expression under the control of a heterologous promoter in L. lactis. Analysis of the homology between the CluA protein and the related proteins of Enterococcus and Streptococcus allowed a common structure for these proteins to be postulated. This consisted of five domains. Functionally conserved domains I and V act respectively as a secretary leader and C‐terminal membrane anchor. Domains II and IV are conserved at the amino acid level and probably have common structural roles whereas domain III is variable and may control binding specificity.
Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum
Authors: Leganés F. , Fernández‐Piñas F. , Wolk C. Peter 
Published: October 2006

SummaryEvident differentiation of vegetative cells into hetero‐cysts in Anabaena sp. strain PCC 7120 is prevented by Insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild‐type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored, in contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.
Effects of an isogenic Zn‐metalloprotease‐deficien mutant of Legionella pneumophila in a guinea‐pig pneumonia model
Authors: Moffat Jennifer F. , Edelstein Paul H. , Regula Donald P. , Cirillo Jeffrey D. , Tompkins Lucy S. 
Published: October 2006

SummaryTo determine the effects, if any, of the Zn‐metallo‐protease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acantha‐moeba cell model, in guinea‐pig macrophages, and in a guinea‐pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin‐resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea‐pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild‐type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea‐pig model of infection employing the intratracheal Inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain.
The ompA 5′ untranslated region impedes a major pathway for mRNA degradation in Escherichia coli
Authors: Hansen Michael J. , Chen Li‐How , Fejzo Mariena L. S. , Beiasco Joel G. 
Published: October 2006

SummaryThe unusual longevity of the Escherichia coli ompA transcript is determined by its 5′untranslated region (UTR), which functions In vivo as an mRNA stabilizer. Here we show that this 5′UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is Joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5′ UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome‐binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5′ UTR is not RNase III.
Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium
Authors: Perez M. Hernandez , Fomukong N. G. , Hellyer T. , Brown I. N. , Dale J. W. 
Published: October 2006

SummaryA highly mobile Insertion sequence designated IS 1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20′) were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS 1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS 1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110‐derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110‐hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited In M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.