The gene-protein database of Escherichia coli: edition 5.

The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.     

The gene-protein database of Escherichia coli: edition 4.

The gene-protein database of Escherichia coli has as its core an index that links each of the protein spots from a two-dimensional polyacrylamide gel to the gene that encodes the protein. Additional information about each protein and its gene is generated from two-dimensional gel analysis or collated from the literature to form the database. Earlier editions of the database have provided periodic updates of information. The current edition does this, but also introduces a new reference gel image produced by an electrophoresis system recently adopted in this laboratory. The new gel system was chosen because it offers an improved opportunity for other investigations to produce close replicas of the reference gel pattern, thereby allowing easier access to the information of the database and encouraging independent contribution to the database. The new gel format also is larger and hence more compatible with computer assisted image analysis, which has become essential for a project of this magnitude. This edition continues the use of the former reference gel images, but adds a reference image of an equilibrium gel of E. coli strain W3110 produced by the new standardized gel system. At this time, 55% of the protein spots annotated on the previous equilibrium reference gel for this organism have been located on the new reference image, and these identifications are included in the tables of the database.     

RECOVERY OF Escherichia coli K-12 FROM NEAR-ULTRAVIOLET RADIATION-INDUCED MEMBRANE DAMAGE

Abstract A wild-type Escherichia coli K-12 strain was irradiated with broad-band near-ultraviolet radiation (from Black-Light Blue fluorescent lamps) and after holding at 37°C for various times in a complex recovery medium, was assessed for viability on either complex medium (YENB) or minimal medium containing a high inorganic salt content. A near-ultraviolet radiation fluence was used which reduced the surviving fraction to approximately 10% when assessing for viability on the complex medium plates. A near-ultraviolet radiation induced sensitivity to inorganic salt was observed which was largely recoverable by holding treated cells in a complex recovery medium. The majority of the recovery process occurred in the initial 2 h post-irradiation holding period. No inhibition of the recovery process was produced by adding chloramphenicol (40 μg/m l ) or penicillin G (11 units/m l ) to the recovery medium, indicating that neither protein synthesis nor cell wall synthesis, respectively, were required for recovery. However, by adding bacitracin, an antibiotic which acts in part by inhibiting membrane synthesis, to the recovery medium, an effect on recovery from salt sensitivity was observed. At the concentrations of bacitracin used (0.6 and 0.2 units/m l ), little or no effect was observed on unirradiated cells, but both concentrations decreased the amount of recovery of irradiated cells. These results demonstrate that recovery from near-ultraviolet radiation-induced salt sensitivity occurs, it is independent of cell growth and the effect of bacitracin suggests that membrane synthesis may be required for recovery.     

AN ACTION SPECTRUM FOR ULTRAVIOLET RADIATION-INDUCED MEMBRANE DAMAGE IN Escherichia coli K-12

A wild-type Escherichia coli K-12 strain was irradiated using monochromatic radiation in the range 254 to 405 nm. A measure of the cell membrane damage induced at each wavelength was investigated by comparing cell viability after irradiation on nutrient agar and on minimal medium containing either a low or high inorganic salt concentration. An action spectrum for lethality and for cell membrane damage was then determined. From 254 to 310 nm lethality closely corresponded to the absorption spectrum of DNA, and there was no indication of membrane damage. However, above a wavelength of 310 nm, the direct absorption of radiation by DNA could not account for the sensitivity observed. Moreover, at wavelengths longer than 310 nm, cell membrane damage was induced and by an increasing factor up to a peak at 334 nm. At the longer wavelengths of 365 and 405 nm, there was a gradual decrease from the peak of damage to cell membranes induced by 334 nm radiation. These results indicate that cell membrane damage may contribute significantly to near-UV radiation-induced cell lethality in wild-type E. coli K-12.     

Photobiological effects of metal ions. Photosensitizing action of uranyl acetate on Escherichia coli K-12

Abstract— The Uranyl acetate sensitized killing of Escherichia coli K-12 by a light source approximating sunlight in both intensity and wavelength distribution is demonstrated to occur at a concentration of 5.0 × 10O^-4M uranyl acetate (pH 7.0). The photosensitized killing was evident after 150 min and almost complete within 320 min. Auxotrophic mutants were isolated from cultures incubated in both light and dark for 160 min at this concentration of uranyl acetate. Binding of UO₂²⁺ to E. coli is shown to occur with 82% of the UO₂²⁺ ions in a 5 × 10^-4M solution (pH 7.0) being bound to the cell wall. In the dark as well as at other pH values the extent of binding was much less. Most of the binding occurred in a time less than 30 min. The observation of rapid binding but delayed photosensitization is attributed to the necessity for penetration of uranyl ions into the cells' interior to effect photosensitization.     

PHOTOREACTIVATION OF ULTRAVIOLET RADIATION DAMAGE IN DARK REPAIR DEFICIENT PHR MUTANTS OF Escherichia coli K-12

There is uncertainty in the literature concerning the genetic control of photoreactivation in E. coli. Two genetic loci, phrA and phrB have been proposed, and two photolyase molecules have been isolated, but in vivo evidence for the activity of the former is controversial. We have studied photoreactivation after 254 nm UV in a dark-repair-deficient phrB mutant and in a strain deleted at the proposed phrA locus. We show apparent photoenzymatic repair in the phrB mutant, which is abolished when the mutation is transduced into the proposed phrA deletion mutant. We conclude that there is a gene in the region of the proposed phrA locus which affects photoenzymatic repair.     

Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock

We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts. Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-[35S]methionine were compared using computing scanning densitometry. The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C. A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested. This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms. For instance, protein SSB was specifically overexpressed by shocked immobilized organisms. Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.     

Expression and initial characterization of WbbI, a putative D-Galf:alpha-D-Glc beta-1,6-galactofuranosyltransferase from Escherichia coli K-12

Cloning of E. coli K-12 orf8 (wbbI) and over-expression of the corresponding enzyme as a maltose-binding fusion protein provided recombinant WbbI beta-1,6-galactofuranosyltransferase activity. Challenged with synthetic acceptor analogues in the presence of UDP-galactofuranose as a donor, WbbI showed a modest preference for pyranoside acceptor substrates of the alpha-D-gluco-configuration but it also possessed the ability to turn-over acceptor analogues.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

PHOTOREACTIVATION IN A phrB MUTANT OF Escherichia coli K-12: EVIDENCE FOR THE ROLE OF A SECOND PROTEIN IN PHOTOREPAIR

Abstract In Escherichia coli , the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phr B, another locus phr A having been proposed in association with PR. The relevance of the putative phr A gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosphila melanogaster . which specifically repairs pyrimidine (6–4) pyrimidone photoproducts ([6–4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6–4) photoproduct, has cast new light on the prospective role of phr A in PR. We have determined the nucleotide sequence of the putative phr A gene, which suggests it codes for a protein of 38 kDa. When the putative phr A gene was cloned into an expression vector and transformed into a phr A phr B mutant of E. coli , a level of photorepair was observed, which could correspond to repair of (6–4) photoproducts.     

Low frequency electric polarizability and zeta-potential of Escherichia coli HB101 (K-12) cells during inactivation with ethanol

The electric properties of bacteria determine their non-specific interactions with the environment, in particular their pathogenic activity. The electric polarizability of Escherichia coli HB101 (K-12 strain) was studied while inactivation with ethanol (20–40 vol.%). The current investigation might be regarded as a continuation of previous research on the polarizability of E. coli at lower ethanol concentration (≤ 20 vol.%) and higher frequencies (≥ 20 kHz). The bacteria polarizability at low frequencies (<10⁴ Hz) shows anomalies (unexpected increase in the polarizability at certain ethanol concentrations), while the parameter decreases with an increase in the ethanol concentration at higher frequencies. We investigated for the possible reasons causing the anomalies — in our case reduced to the medium dielectric permittivity, the average cell length and the surface electric charge density distribution, related to bacterial lipopolysaccharides. We suggest a hypothesis for the molecular mechanism of changing the surface charge of E. coli, carried by lipopolysaccharides, induced by the non-ionic ethanol.      

Photoreactivation in phr mutants of Escherichia coli K-12: cloning from the gal-att lambda interval increases the photoreactivable response in phrA strains

In this report we have cloned restriction fragments from the gal-att lambda region obtained from a purified preparation of lambda dgal transducing 'phage DNA, and demonstrate the appearance of a photoreactivable response in a photoreactivation-deficient phrA phrB strain. We also show that when this plasmid is transduced into a delta phrA strain there is an increase in the photoreactivable response after a single high intensity light flash and after continuous illumination. These data have been discussed in relation to the hypothesis of the presence of multiple photolyase molecules in Escherichia coli.     

ROLES OF URACIL-DNA GLYCOSYLASE AND APYRIMIDINIC ENDONUCLEASES IN THE MOLECULAR 5-BROMO-2'-DEOXYURIDINE PHOTOSENSITIZATION IN Escherichia coli K-12

The molecular mechanism for 5-bromo-2'-deoxyuridine (BrdU) photosensitization was studied in thymine-requiring wild-type and uracil-DNA glycosylase (UDG)-deficient ung mutant cells of Escherichia coli K-12. Wild-type cells were more sensitive to BrdU photosensitiLation than ung mutant cells. IJV induced the identica/ numbers of alkaline sucrose single-strand breaks (SSB) in 5-bromouracil-DNA (BrU-DNA) of both the wild type and ung mutant. The ung mutant cells repaired SSB almost completely, whereas the wild-type cells with UDG produced more adverse SSB by 90 min after UV. Neutral agarose gel electrophoresis of minipreps indicated that UV induced (1) more smears of host BrU-DNA possibly by more double-strand breaks (DSB) and (2) a greater decline of pBR322 Form I BrU-DNA in the wild-type cells than the ung cells. These results indicated a greater induction of SSB by apyrimidinic (AP) endonucleases in wild-type cells. The ung/ wild ratios (=1.7–1.9) for cellular and plasmid BrdU sensitizations aftcr growth in 50% BrdU were similar. The extents of UDG-dependent and UDG-independent sensitizations in wild-type cells were ∼40 and ∼60%, respectively. The xth nfo double mutant defective in both exonuclcase III and endonucleasc IV was more sensitive to BrdU photosensitization than the wild type, indicating that an excess of AP sites remaining after uracil excision in the xth nfo mutant causes a greater BrdU photosensitization than SSB by AP endonucleases in wild-type cells. Conversely, the xth rfo ung triple mutant was more resistant to BrdU photosensitization than the xth nfo double mutant, so that UV-induced uracil residues in the BrU-DNA are tolerated and do not appear to be directly responsible for BrdU photosensitization.     

Human cerebrospinal fluid protein database: edition 1992.

Two-dimensional electrophoresis maps of human cerebrospinal fluid proteins are presented in the form of labeled images. 931 protein spots are identified in spinal fluid from a normal volunteer. Distinct spots that represent variants of the same protein, especially posttranslational modifications, are estimated to reduce the 931 different spots to < 200 different proteins. 248 spots of 29 protein groups have been identified and are indicated on enlargements of specific gel regions. The distribution of protein abundance, mass, charge and shape characteristics of these normal 931 spinal fluid spots are graphically profiled. Analysis of the shape parameter "vertical height: width ratio" reveals that a ratio > 3.5 correlates with glycoproteins, enabling their identification simply by image analysis. Proteins that are not present on the normal map, but appear in spinal fluid in patients with schizophrenia and Creutzfeldt-Jakob disease are illustrated on additional maps.     

SENSITIZATION OF ULTRAVIOLET-IRRADIATED Escherichia coli K-12 BY DIFFERENT AGARS: INHIBITION OF A rec AND exr GENE-DEPENDENT BRANCH OF THE uvr GENE-DEPENDENT EXCISION-REPAIR PROCESS

Abstract— A plaque assay for adenovirus 2 on normal human fibroblasts has been developed and used to measure the survival of ultraviolet-irradiated virus on six human fibroblast cell lines. When four xeroderma pigmentosum cell lines were used as viral hosts, an average of one lethal event per virus in the viral population was made with 10, 15, 62, and 78 J m^-2 respectively, while using two normal cell lines as hosts, 197 and 205 J m^-2 were required to inflict the same damage. These differences are attributed to the known repair deficiency of xeroderma pigmentosum cells, and are discussed in the light of previous data obtained using other animal viruses.