bluuebunny/crossref_metadata_2025_split huggingface dataset:
Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate‐forming enzymes
Authors: Turgay K. , Krause M. , Marahiel M. A. 
Published: October 2006

SummaryThe entire nucleotide sequence of the Bacillus brevis grsB gene encoding the gramicidin S synthetase 2, which activates and condenses the four amino acids proline, valine, ornithine and leucine has been determined. The gene contains an open reading frame of 13359bp which encodes a protein of 4453 amino acids with a predicted Mr of 510287. The gene is located within the gramicidin S biosynthetic operon, also containing the genes grsT and grsA, whose nucleotide sequences have been determined previously. Within the GrsB amino acid sequence four conserved and repeated domains of about 600 amino acids (45–50% identity) have been identified. The four domains are separated by non‐homologous sequences of about 500 amino acids. The domains also share a high degree of similarity (20–70%) with eight peptide synthetases of bacterial and fungal origin as well as with conserved sequences of nine other adenylate‐forming enzymes of diverse origin. On the basis of sequence homology and functional similarities, we infer that those enzymes share a common evolutionary origin and present a phylogenetic tree for this superfamily of domain‐bearing enzymes.
Outer membrane protein P6 of Haemophilus influenzae binds to its own gene
Authors: Sikkema Daniel J. , Nelson M. Bud , Apicella Michael A. , Murphy Timothy F. 
Published: October 2006

SummaryOuter membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix‐turn‐helix DNA‐binding proteins. A search for sequence‐specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6‐specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleo‐tide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence‐specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram‐negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.
A theoretical and experimental analysis of bacterial growth in the bladder
Authors: Gordon David M. , Riley Margaret A. 
Published: October 2006

SummaryA mathematical model of human micturition dynamics and bacterial growth predicts the population growth rate required for a bladder infection to become established in the absence of adhesin‐mediated surface growth. Escherichia coli strains isolated from the urinary tract have significantly higher in vitro growth rates in urine than strains isolated from the intestinal flora. The results suggest that, for E. coli isolated from the urinary tract, adhesin‐mediated surface growth may not be required for infections to become established and persist. The growth‐rate differences observed between urinary tract and Intestinal isolates suggests that the ability to survive and efficiently utilize the resources available in urine is an important adaptation for E. coli inhabiting the urinary tract.
DNA in dormant spores of Bacillus species is in an A‐like conformation
Authors: Setlow Peter 
Published: October 2006

SummaryThe DNA in dormant spores of Bacillus species is associated with α/β‐type small, acid‐soluble proteins (SASP), which are double‐stranded DNA‐binding proteins whose amino acid sequence has been highly conserved in evolution. In vitro these proteins bind most strongly to DNA which readily adopts an A‐like conformation, as binding of α/β‐type SASP causes DNA to assume an A‐like conformation. As predicted by this conformational change in DNA, binding of α/β‐type SASP to relaxed but covalently closed plasmid DNA results in the introduction of a large number of negative supercoils. Associated with the conformational change in DNA brought about by α/β‐type SASP binding is a change in its photochemistry such that ultraviolet irradiation does not generate pyrimidine dimers, but rather a thyminyl‐thymine adduct termed spore photoproduct (SP). The latter two properties of DNA complexed with α/β‐type SASP in vitro are similar to those of DNA in dormant spores of Bacillus species in which: (i) plasmid DNA has a much higher number of negative supercoils than plasmid in growing cells; and (ii) ultraviolet irradiation produces SP and no pyrimidine dimers, while only pyrimidine dimers are formed in growing cells. During sporulation these changes in the properties of spore DNA take place in parallel with synthesis of α/β‐type SASP, and the magnitude of the changes is greatly reduced in mutants that make low amounts of these proteins. A straightforward interpretation of these data is that DNA in dormant spores of Bacillus species is in an A‐like conformation. DNA in an A‐like conformation is also present in growing cells with high α/β‐type SASP levels. However, the presence of this A‐like DNA is detrimental to cell growth.
Z‐DNA as a probe for localized supercoiling in vivo
Authors: Rahmouni A. Rachid 
Published: October 2006

SummaryBiological processes such as transcription are expected to generate local variations in DNA super‐coiling. The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil‐driven B‐to‐Z transition as a super‐helicity probe. This new methodology is described and its extension to other biological systems discussed.
Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region
Authors: Krohn M. , Pardon B. , Wagner R. 
Published: October 2006

SummaryTranscription elongation catalysed by DNA‐dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid‐encoded ribosomal RNA operon. Plasmid templates with different super‐helical densities (σ between +0.0017 and ‐0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.
Tumour necrosis factor alpha antibody protects against lethal meningococcaemia
Authors: Nassif X. , Mathison J. C. , Wolfson E. , Koziol J. A. , Ulevitch R. J. , Son M. 
Published: October 2006

SummaryTumour necrosis factor alpha (TNF‐α) has been shown to be the principal mediator of Gram‐negative bacterial endotoxin‐induced shock. Nevertheless, evidence suggests that TNF‐α plays a beneficial role in controlling bacterial infections when multiplication of the microorganism is required to kill the host. Using an infant rat model of Neisseria meningitidis infection, we found that blood TNF‐α concentration reaches a peak three hours after intraperitoneal injection of 3 × 106 bacteria. Thereafter, the level of TNF‐α decreased and was undetectable six to eight hours after infection. A correlation was observed between the magnitude of initial TNF‐α response and a fatal outcome. Pretreatment of the animals with polyclonal anti‐TNF antiserum significantly reduced mortality relative to animals pretreated with control serum. However, pretreatment of animals with anti‐TNF antibody did not alter the bacterial invasion of the cere‐brospinal fluid. Injection of heat‐killed bacteria did not cause death and induced lower TNF‐α levels than the same number of live bacteria. This excludes the possibility that the role of TNF‐α is to mediate a shock induced by the endotoxin component of the bacterial inoculum. These results indicate that TNF‐α has a deleterious effect in this model of bacteraemia. Identification of the critical factors that determine the action of TNF‐α during lethal bacteraemia will lead to a better understanding of these diseases and the development of appropriate therapeutic intervention.
SecB‐binding does not maintain the translocation‐competent state of prePhoE
Authors: de Cock Hans , Tommassen Jan 
Published: October 2006

SummaryThe rote of SecB protein in the export of the precursor of outer membrane protein PhoE and mutant forms of this precursor was studied in vitro. When synthesized in the absence of SecB, translocation‐competent prePhoE was observed post‐translationally, but addition of SecB was required for efficient translocation into inner membrane vesicles. The translocation competency of in vitro synthesized prePhoE diminished with a similar half‐life during incubations in the presence or absence of SecB. The loss of translocation competency of prePhoE, synthesized in the presence of SecB, was not due to dissociation of prePhoE–SecB complexes as could be demonstrated in co‐immunoprecipitation experiments with anti‐SecB serum. Apparently, SecB does not maintain the translocation‐competent conformation of prePhoE, but is mainly required for efficient targeting of this precursor to the export apparatus.
Regulation of the expression of the cell‐cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules
Authors: Tétart Françoise , Bouché Jean‐Pierre 
Published: October 2006

SummaryWe show that the 53‐nucieotide RNA molecule encoded by gene dicF blocks cell division In Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome‐binding region of the ffsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ–lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.
Genes and their organization in the replication origin region of the bacterial chromosome
Authors: Ogasawara Naotake , Yoshikawa Hiroshi 
Published: October 2006

SummaryGenes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram‐positive and Gram‐negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram‐positive bacteria were diverged from the Gram‐negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non‐translatable region containing multiple repeats of DnaA‐box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.
Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3‐type cytochrome c oxidase of Rhodobacter sphaeroides
Authors: Shapleigh James P. , Gennis Robert B. 
Published: October 2006

SummaryThe ctaD gene encoding subunit I of the aa3‐type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild‐type strain owing to the presence of an alternate o‐type cytochrome c oxidase. The aa3‐type oxidase was restored by complementing the chromosomal deletion with a plasmid‐borne copy of the CtaD gene. This system is well suited for site‐directed mutagenesis probing of the structure and function of cytochrome c oxidase.
Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene
Authors: Chen Yi‐Chi , Shipley Gregory L. , Ball Timothy K. , Benedik Michael J. 
Published: October 2006

SummaryThe extracellular nuclease of Serratia marcescens is regulated in a complex fashion. Unlike most catabolic enzymes, it appears not to be substrate regulated. However we have shown it to be regulated by an SOS‐like system in S. marcescens. Additionally nuclease expression is regulated In a growth‐phase‐dependent manner. In this work we demonstrate that growth‐phase‐dependent regulation is at the transcriptional level. The putative LexA‐binding site which mediates SOS regulation is shown to act as an operator site in viva. The boundaries of a minimal promoter, stilt regulated by growth phase and SOS regulation, are defined along with the transcriptional start site. However, a region upstream of the nuclease promoter is shown to enhance significantly the expression of nuclease.
Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities
Authors: Srikumar R. , Chin A. C. , Vachon V. , Richardson C. D. , Ratcliffe M. J. H. , Saarinen L. , Käyhty H. , Mäkelä P. H. , Coulton J. W. 
Published: October 2006

SummaryThe major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial ceil surface, seven mouse monoclonal anti‐Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104–139; domain (ii) amino acids 162–174; and domain (iii), amino acids 267–341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement‐mediated tysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface‐binding, they were neither bactericidal nor protective.
Microtubule assembly and phage morphogenesis: new results and classical paradigms
Authors: Weinstein Brant , Solomon Frank 
Published: October 2006

SummaryThe classical analyses of phage morphogenesis provide experimental paradigms for dissecting other assembly pathways. A new set of results, derived from examination of the quantitative controls of tubulin levels and microtubule assembly in Saccharomyces cerevisiae, evokes the balance of components’ hypothesis. These results show that balanced levels of the tubulin proteins are crucial for microtubule assembly. Imbalances leading to excess beta tubulin have far more deleterious consequences than those leading to excess alpha tubulin, including dramatic cellular toxicity, quantitative depolymerization of cellular microtubules, and self‐aggregation of the excess beta tubulin. These and other results suggest that beta tubulin may possess a unique ability to interact with a component of microtubule nucleating sites, and provide a rationale for the universal polarity of nucleated microtubules.
Elongation factor Tu: a molecular switch in protein biosynthesis
Authors: Weijland Albert , Harmark Kim , Cool Robbert H. , Anborgh Pieter H. , Parmeggiani Andrea 
Published: October 2006

SummaryElongation factor Tu (EF‐Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide‐binding protein that in the ‘on’ state acts as a carrier of amino acyl‐tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co‐ordinated application of biochemical, physico‐chemical and genetic methods. Crystallographic analysis at 2.6 Å resolution and site‐directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide‐binding domains of EF‐Tu and human H‐ras p21 protein. The regulation of the expression of the two EF‐Tu‐encoding genes in E. coli, particularly that of tufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF‐Tu in the elongation cycle have been reinvestigated, leading to new insights. These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF‐Tu in the bacterial cell.
Establishment of cell‐specific transcription during sporulation in Bacillus subtilis
Authors: Errington J. , Illing N. 
Published: October 2006

SummaryOne of the most intriguing questions posed by bacterial spore formation concerns the establishment of cell‐specific gene expression in the prespore and mother cell. Recent results now suggest that sigma factors, in addition to their temporal roles in the control of gene expression, may also be the key determinants of differential gene expression during sporulation in Bacillus subtilis. The genes encoding two sporulation‐specific sigma factors, σE and σF, are expressed soon after the initiation of sporulation, before the formation of the spore septum that separates the prespore and mother cell compartments, it now appears that σE and σF direct transcription only after septation and then In a specific cell type, suggesting that the segregation of the sigma activities after septation is a key event in the establishment of differential gene expression. The mechanism responsible for this segregation is complex, involving at least seven other gene products. We discuss possible models for the interactions between the sigma factors and the establishment of cell‐specific transcription.
A key role for type 1 pili in enterobacterial communicability
Authors: Bloch Craig A. , Stocker Bruce A. D. , Orndorff Paul E. 
Published: October 2006

SummaryUp to 80% of faecal Escherichia coil strains are able to produce type 1 pili. These filamentous bacterial surface organelles, which mediate mannose‐sensitive attachment to mammalian epithelial cells, are also conserved throughout the Enterobacteriaceae. As a potential explanation for their prevalence among intestinal isolates of enteric bacteria, it has been widely speculated that type 1 pili are important for adherence to the host's intestinal mucosa. However, conclusive evidence for this idea is lacking, and there are reasonable grounds for doubting such an effect. Permanent interruption of type 1 pillation in previously pil+E. coli (by directed mutagenesis of pilA, the gene coding for the major structural subunit of type 1 pili) does not diminish the density of intestinal colonization in individual animals. Rather, as we demonstrate here, this lesion results in a dramatic decrease in transmission of E. coli K1 from experimentally colonized neonatal rats to their littermates. The enhanced communicability associated with type 1 pillation suggests a heretofore unrecognized explanation for the prevalence of type 1 pili among intestinal E. coli; one that does not necessarily require the direct action of these organelles at the intestinal mucosa.
An amino‐proximal domain required for the localization of FtsQ in the cytoplasmic membrane, and for its biological function in Escherichia coli
Authors: Dopazo Ana , Palaclos Pilar , Sánchez Manuel , Pla Jesús , Vicente Miguel 
Published: October 2006

SummaryThe location of FtsQ, an Escherichia coli protein essential for cell division, is, under physiological conditions, in the cytoplasmic membrane facing towards the periplasmic space. An amino‐proximal hydrophobic domain is required for FtsQ to reach its location and for its activity in the cell. Overexpression of modified forms of FtsQ is deleterious for the cell.
The interaction of naturally elaborated blebs from serum‐susceptible and serum‐resistant strains of Neisseria gonorrhoeae with normal human serum
Authors: Pettit Robin K. , Judd Ralph C. 
Published: October 2006

SummaryWe studied the interaction of normal human serum immunoglobulins with outer‐membrane bleb antigens of Neisseria gonorrhoeae. Gonococcal 68000 Dalton and Lip (H.8 antigen) outer‐membrane proteins were recognized by normal human serum immunoglobulins in blebs from serum‐resistant strains, but not in blebs from serum‐susceptible strains. The addition of blebs from a serum‐resistant strain to bactericidal assays resulted in significantly greater inhibition of serum killing than the addition of blebs from a serum‐susceptible strain. Our results indicate that blebs from two serum‐resistant gonococcal strains have an enhanced ability to bind and remove cell‐targeted bactericidal factors, and that outer‐membrane bleb‐bing may contribute to serum resistance.