DNA- and RNA-binding proteins of chromatin from Escherichia coli

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sj?stad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.     

Resistance to quaternary ammonium compounds in Staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pST827

K.A. ALBASHERI AND W.J. MITCHELL. 1995. Maltose metabolism in the obligate anaerobe Clostridium acetobutylicum was studied. The sugar is accumulated via an energy-dependent transport process which is not a phosphotransferase. Cell extracts were incapable of phosphorylating maltose in the presence or absence of phosphoenolpyruvate or ATP, but exhibited hydrolytic activity against a range of glucoside substrates. The activity was predominantly in the soluble fraction of cell extracts, indicating a cytoplasmic location in the cell. Gel filtration on Sephadex G100 indicated the presence of at least two α-glucosidases. One enzyme (maltase) was active with maltose and maltotriose, while the other (pNPGase) hydrolysed isomaltose and several glucoside analogues, but neither showed activity against starch. Both glucosidases were induced by isomaltose, maltose, glucose and starch, but not by xylose, sucrose or cellobiose. In the presence of both glucose and maltose, growing cells showed a preference for glucose, apparently due to regulation of maltose transport, which did not occur in glucose-grown cells.     

Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide.

Preliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB-37, Shiva-1, and Vishnu), and enhanced proliferation was determined by uptake of 3H-thymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 microM or less SB-37. The primary study consisted of 263 four-cell- to eight-cell-stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 microM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.     

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.     

The qacG gene on plasmid pST94 confers resistance to quaternary ammonium compounds in staphylococci isolated from the food industry

The 2.3 kb resistance plasmid pST94 revealed a new gene (qacG) encoding resistance to benzalkonium chloride (BC), a commonly used quaternary ammonium disinfectant, and the intercalating dye ethidium bromide (Eb) in staphylococci isolated from the food industry. The 107 amino acid QacG protein showing 69.2% identity to the staphylococcal multi-drug resistance protein Smr is a new member of the small multi-drug resistance (SMR) protein family. QacG conferred resistance via proton dependent efflux. An additional ORF on pST94 encoded a protein with extensive similarity to replication proteins of other Gram-positive bacteria. Gene constructs containing the qacG and smr gene region combined with the smr or qacG promoter, respectively, indicated that QacG is more efficient than Smr and that qacG has a weaker promoter. Resistant qacG-containing cells could be adapted to withstand higher concentrations of BC. Adapted qacG-containing cells showed increased resistance mainly to BC. In contrast, adaptation of sensitive cells showed cross-resistance development to a range of compounds. Induction of proton-dependent efflux was observed for BC-adapted staphylococci cells not containing qacG. The ability of sublethal concentrations of BC to develop cross-resistance and induce efflux mechanisms could be of practical significance; it should be considered before use of any new disinfectant and in the design of better disinfection procedures.     

Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers

AIMS: To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross-resistance to antibiotics in Escherichia coli. METHODS AND RESULTS: Precultivation of E. coli ATCC 11775 and E. coli DSM 682 in the presence of subinhibitory concentrations of BC or stress inducers (salicylate, chenodeoxycholate and methyl viologen) resulted in higher minimum inhibitory concentration (MIC) of BC and chloramphenicol (CHL). Adaptation to growth in sixfold of the initial MIC of BC resulted in stable BC resistance and enhanced tolerance to several antibiotics and ethidium bromide (EtBr). The MIC of CHL increased more than 10-fold for both strains. Enhanced efflux of EtBr in adapted E. coli ATCC 11775 indicated that the observed resistance was due to efflux. Changes in outer membrane protein profiles were detected in the BC-adapted cells. There were no indications of lower membrane permeability to BC. CONCLUSIONS: Induction of stress response or gradual adaptation to BC or CHL results in acquired cross-tolerance between BC and antibiotics in E. coli. Enhanced efflux was one of the observed differences in adapted cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided not taking due precautions, extensive use of disinfectants could lead to emergence of antibiotic-resistant isolates.     

Gastric inflammatory markers and interleukins in patients with functional dyspepsia treated with astaxanthin

The chronic active inflammation caused by Helicobacter pylori is dominated by neutrophils, macrophages, lymphocytes and plasma cells. Several interleukins are involved in the inflammatory process. The aim of this study was to investigate the effect of astaxanthin on gastric inflammation in patients with functional dyspepsia. Forty-four consecutive patients were included, and biopsies were examined for IL-4, IL-6, IL-8, IL-10, interferon-gamma, CD4, CD8, CD14, CD19, CD25 and CD30. Patients were randomized: 21 patients were treated with 40 mg of astaxanthin daily, and 23 patients were treated with a placebo. There was a significant decrease in gastric inflammation in H. pylori-positive patients from both groups. There were no significant changes in the density of H. pylori or in any of the interleukins during or after treatment. There was a significant up-regulation of CD4 and down-regulation of CD8 in patients with H. pylori treated with astaxanthin. Astaxanthin had an effect on the inflammation and on the density of H. pylori in mice in a study where the diet could be standardized without antioxidants (Bennedsen et al., 1999). These dietary conditions are impossible in studies involving humans, and may be due to the minor effect when the host have access to antioxidants in their diet.     

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.