The electrooptical parameters of suspensions of <Emphasis Type="Italic">Escherichia coli</Emphasis> XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 198–203.Original Russian Text Copyright © 2005 by Bunin, Ignatov, Guliy, Zaitseva, ONeil, Ivnitskii     

The effect of ampicillin on the electrophysical properties of Escherichia coli cells

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OS) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10-1000 kHz of the orienting electric field and does not affect the OS of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 microg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OS of cells can be used to evaluate their resistance to ampicillin.     

Sensitivity of various <Emphasis Type="Italic">Escherichia coli</Emphasis> strains to 2,4,6-trinitrotoluene

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100–200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 53–57.Original Russian Text Copyright © 2005 by Kurinenko, Denivarova, Yakovleva.     

A correlation between sensitization to gamma-rays by purine starvation and the rec genes in Escherichia coli K-12

Summary After purine staration the recombination proficient (Rec⁺) strains of E. coli K-12 tested were sensitized to inactivation by gamma-rays (Figs. 1–4). No such change was noted in the Rec^– strains tested (Fig. 5). These results suggest that purine starvation may sensitize cells to ionizing radiation by inhibiting repair controlled by the recA, recB and recC genes.This paper was reported in part at the Fourteenth Annual meeting of the Biophysical Society in Baltimore, Md., 25–27 February 1970.     

Plasmid mediated silver resistance in Acinetobacter baumannii

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10^–6 recepient^–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10^–8 recepient^–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

The chemotactic characteristics of the S and R dissociants of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The chemotactic responses of Bacillus thuringiensis subsp. dendrolimus (strain 49) and thuringiensis (strain 2002) and their morphological dissociants were studied by using some natural and artificial substances as effectors. The 12-h-old wild-type cells (S variants) of both strains were found to be motile and similar in their chemotactic responses, whereas the chemotactic responses of the R variants were different.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 87–91.Original Russian Text Copyright © 2005 by Lebenko, Sekerina, Chemerilova.     

Survival, mutation and capacity to repair single-strand DNA breaks after gamma irradiation in different Exr? strains of Escherichia coli

Summary Strains of Escherichia coli K-12 and B/r made by transduction of the exrA allele from a B_s-2 derivative have been compared with Exr(W)^– strains derived from B_s-1 and B_s-2 by mutation (E.M. Witkin, 1967). Both transduced exrA and Exr(W)^– strains were almost unmutable by gamma radiation, but the former class were as sensitive to gamma radiation as recA strains and, like them, were unable to repair single-strand DNA breaks as detected by the McGrath-Williams technique. In contrast the Exr(W)^–strains were as resistant to gammaradiation as Exr(W)⁺ strains derived from them and were equally efficient in repairing single-strand breaks. The existence of Exr(W)^–strains suggests that the mutagenicity of single-strand breaks may depend entirely on the way in which they are repaired. The properties of the (Exr(W)^–strains cannot be ascribed solely to the transducable exrA allele.A large effect of diffuse daylight in lowering the molecular weight of DNA on alkaline sucrose gradients is described which, unless prevented, may lead to erroneous results in such experiments.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

The production of recombinant beta-galactosidase inEscherichia coli in yeast extract enriched medium

Summary The productivity ofEscherichia coli biomass and recombinant beta-galactosidase was increased in Luria broth (LB) enriched with yeast extract. In flask culture under conditions of LB limitation, yeast extract suplementation gave the highest biomass (strain HB101/pRW756) stimulation per unit of component added compared with supplementation by various amounts of amino acids, vitamins, minerals, purines/pyrimidines, tryptone, casamino acids, casein peptone or gelatin peptone. The biomass production ofE. coli HB101/pRW756, XL-1 blue/puc118, XL-1 Blue FF/puc118 and TB-1/p1034 cells was stimulated in fermentor-scale experiments with additional yeast extract in LB. Total beta-galactosidase production from plasmid genes in fermentor-scale experiments was increased 105.4% in XL-1 blue/puc118 cells, 365.5% in XL-1 blue FF/puc118 cells and 421.4% in TB-1/p1034 cells by 0.5%, 1% and 1% weight per volume of additional yeast extract in LB, respectively. Depending on different strains, the increase of the enzyme production was obtained either by increased biomass, or the combination of enhanced gene expression and increased biomass. Neither the biomass nor beta-galactosidase production was stimulated in N4830/p1034 cells by the increase in yeast extract concentration in the medium.     

Cloning of the Genes That Control Formation of the Fimbrial Colonization Factor Antigen III (CFA/III) from an Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strain 260-1 produces colonization factor antigen III and heat-labile enterotoxin. A 55-kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K-12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260-1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4-kb region was found to be responsible for the expression of the pili.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Potential for gene transfer from recombinantEscherichia coli K-12 used in bovine somatotropin production to indigenous bacteria in river water

Summary This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), fromEscherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×10⁷ CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated withE. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated withE. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli

Summary To measure the degree of phr gene induction by DNA-damaging agents, the promoter region was fused to the coding region of the lacZ gene in plasmid pMC1403. The new plasmids were introduced into Escherichia coli cells having different repair capabilities. More efficient induction of phr gene expression was detected in a uvrA ^– strain as compared with the wild-type strain. In addition, obvious induction was detected in uvrA ^– cells treated by 4-nitroquinoline 1-oxide and mitomycin C. Nalidixic acid, an inhibitor of DNA gyrase, also induced phr gene expression. In contrast, little induced gene expression was noted in UV-irradiated lexA ^– and recA ^– strains. It is suggested from these results that induction of the phr gene is one of the SOS responses. Possible nucleotide sequences which could be considered to constitute an SOS box were found at the regulator region of the phr gene.Abbreviations phr photoreactivation - UV ultraviolet light - 4NQO 4-nitroquinoline 1-oxide - MMC mitomycin C - PRE photoreactivating enzyme - E. coli Escherichia coli     

Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.