Enhanced survival of plasmid-containing Escherichia coli in aquatic microcosms

The survival of Escherichia coli K-12 J62-1 containing the antibiotic-resistance plasmid R1 and an isogenic plasmid-free strain were studied in pond water microcosms. The number of plasmid-containing cells recovered from the microcosms remained constant over a sampling period of 31 days whereas plasmid-free cell numbers declined.     

Mechanical disruption of Escherichia coli for plasmid recovery

Five different mechanical cell disruption processes were evaluated as methods to extract plasmids from bacterial cells. The methods used were sonication, nebulization homogenization, microfluidization, and bead milling. The recovery yields of intact plasmids from the various methods were measured by quantitative gel electrophoresis. Bead milling and microfluidization were found to have the highest potential for large scale extraction with total intact recoveries of over 90% and around 50%, respectively. Other methods resulted in substantial plasmid degradation, with recoveries no greater than 20% of the total intact plasmid. (c) 1995 John Wiley & Sons, Inc.     

Aerobactin production and plasmid distribution in Escherichia coli clinical isolates

The distribution of plasmids as a function of aerobactin production, antibiotic resistance and antimicrobial agents production was studied in 139 Escherichia coli strains obtained from clinical sources. Ninety eight per cent of the strains analyzed presented plasmids with a median value of 2.97 plasmids per cell. Differences in the number of plasmids were observed for aerobactin production (3.52 for aerobactin producing strains, 2.56 (for non-producing ones) and antibiotic resistance (3.19 for antibiotic resistant strains and 2.58 for the sensitive ones). But this was not the case for antibacterial agent production (2.96 for the producing strains, 2.98 for the non-producing ones. Ecological implications of these results are discussed.     

Transformation and expression of a staphylococcal plasmid in Escherichia coli

Abstract A multiple antibiotic-resistant Staphylococcus aureus , was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one Eco RI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA⁻ E. coli at a frequency of 1.2×10⁴ transformants/μg of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus .     

Effect of growth conditions on the rate of loss of the plasmid pAT153 from continuous cultures of Escherichia coli HB101

Abstract The plasmid pAT153 was lost less rapidly from carbon, nitrogen, phosphorous or sulphur-limited continuous cultures of Escherichia coli HB101 as the dilution rate increased. At a fixed dilution rate of 0.3 h⁻¹, the plasmid was maintained longer as the growth-limiting nutrient was changed from glucose to casamino acids (nitrogen-limited), phosphate or sulphate. These differences in the stability of maintenance were not due to parallel changes in the plasmid copy number. We propose that the rate of loss of pAT153 from E. coli HB101 is determined primarily by the ratio of growth rates of plasmid-containing bacteria and plasmid-free bacteria. This ratio increases with increasing growth rate and depends markedly on the growth-limiting nutrient, sulphate-limited growth being particularly suitable for the maintenance of this host-plasmid combination.     

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli

ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.     

Use of microcosms to determine persistence of Escherichia coli in recreational coastal water and sediment and validation with in situ measurements

AIMS: To determine the persistence of the faecal indicator organism Escherichia coli in recreational coastal water and sediment using laboratory-based microcosms and validation with in situ measurements. METHODS AND RESULTS: Intact sediment cores were taken from three distinct coastal sites. Overlying estuarine water was inoculated with known concentrations of E. coli and decay rates from both overlying water and sediment were determined following enumeration by the membrane filtration method at fixed time intervals over a 28-day period. It was demonstrated that E. coli may persist in coastal sediment for >28 days when incubated at 10 degrees C. Escherichia coli survival was found to have an inverse relationship with temperature in both water and sediment. In general the decay rate for E. coli was greater in water than in sediment. Small particle size and high organic carbon content were found to enhance E. coli survival in coastal sediments in the microcosms. CONCLUSIONS: Results of this microcosm study demonstrated the more prolonged survival of E. coli in coastal sediments compared with overlying water, which may imply an increased risk of exposure because of the possible resuspension of pathogenic micro-organisms during natural turbulence or human recreational activity. SIGNIFICANCE AND IMPACT OF THE STUDY: A more accurate estimate of exposure risk has been described which may subsequently be used in a quantitative microbial risk assessment for recreational coastal waters.     

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure

AIMS: The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. METHODS AND RESULTS: A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. CONCLUSIONS: These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. SIGNIFICANCE AND IMPACT OF THE STUDY: Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.     

Cloning and expression of formaldehyde resistance from Escherichia coli

Abstract Formaldehyde resistance in Escherichia coli strain VU3695 is mediated by a 94 kilobase plasmid. The genes responsible for formaldehyde resistance were identified on a 9.2 kb DNA fragment and cloned in pBR322. By minicell analysis three proteins were shown to be encoded by this fragment.     

Amino acid supplementation decreases plasmid retention in Escherichia coli

The effect of amino acid supplementation on plasmid stability in Escherichia coli B/r was tested experimentally. Comparisons of experimental results to computer-predicted values were made using a detailed, structured single-cell model. The plasmid, pDW17 (a pBR322 derivative with a mutated tac promoter controlling the beta-lactamase gene), was used. In chemostat cultures, the amino acid supplemented cultures were always less stable than those grown in minimal medium. This effect was not a growth rate effect, as increasing growth rate imsproves stability for both cultures in minimal medium and in amino acid supplemented medium. The computer model also predicted a decrease in stability due to amino acid supplementation. The model also predicts that amino acid supplementation, combined with moderately strong plasmid-encoded protein expresion, results in a depletion of low-molecular-weight organics compared with plasmid-free cells. In minimal medium the same level of plasmid-encoded protein synthesis results in a strong reduction in amino acid pools compared with plasmid-free cells. With amino acid supplementation the growth differential between plasmid-bearing and plasmid-free cells may be due to an "energy limitation," while in minimal medium the size of the growth rate differential may be due to a "building block" limitation. (c) 1992 John Wiley & Sons, Inc.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

重组大肠杆菌高密度培养

重组大肠杆菌的高密度培养是增加单位时间,体积重组蛋白产率的最有效途径之一。如何在获得高密度的同时取得较高的单位时间／体积目的蛋白产率,是高密度培养（过程中亟待解决的问题,这与所选用的菌体、构建的表达系统、发酵时pH、溶氧、培养基成分及培养温度、质粒稳定性、代谢副产物的限制及时比生长速率的控制等因素有关。试从这些方面加以综述,分析这些条件对重组蛋白生产的影响,介绍大肠杆菌高密度培养领域的一些研究进展。     

Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coli

The putative gene coding for GTP cyclohydrolase I of Escherichia coli was isolated from a lambda gt11 expression vector library by using antibodies as a probe and has been subcloned on a 3.8 kb Bam HI fragment in the plasmid vector pUC13. E. coli cells carrying the recombinant plasmid designated pCYH express 100-fold increased levels of the enzyme. The protein formed under the control of the plasmid appears electrophoretically and immunochemically identical with the wild type enzyme.     

Decreased cupric ion uptake as the mechanism for cupric ion resistance in Escherichia coli

Abstract Plasmid-encoded copper (Cu²⁺) resistance in Escherichia coli was due to decreased uptake of Cu²⁺. The Cu²⁺-resistant E. coli Rtsl strain contained a 60 MDa plasmid which is known to encode for both Cu²⁺ and kanamycin resistance. A plasmid-free derivative of the same organism exhibited a greater uptake of Cu²⁺, and sensitivity to Cu²⁺ in both respiration and growth studies than the E. coli Rtsl strain.     

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025.H42

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.