Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli

Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.     

Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli.

A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium). Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery. The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay. Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up. Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay. These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted.     

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp.

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F- lambda- wild-type strain, is shown here to carry a cryptic mutation, ftsR1, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42 degrees C and the inability to grow in salt-free LB broth at 42 degrees C. The ftsR1 mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201 (Mecr) and alaS21 (Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a plac-relA' plasmid. These backgrounds also confer resistance in LB broth to the beta-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsR1 mutant (but not an isogenic ftsR+ strain) is sensitive to mecillinam in minimal glucose medium at 37 degrees C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsR1 mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37 degrees C but not the growth defect in salt-free LB broth at 42 degrees C. Genetic analysis indicated that the full phenotype of the ftsR1 mutant is due to a single mutation in the rpoB gene (90 min), coding for the beta subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Adhering ability of Stenotrophomonas maltophilia is dependent on growth conditions

The growth conditions are known to influence the bacterial adhesion to different kinds of surfaces. In the present study the adhering ability of Stenotrophomonas maltophilia, on growth in nutrient rich media (Tryptic Soy Broth (TSB)) and minimal media (Luria Bertani (LB)) was checked by viable cell count and spectrophotometric method. TSB grown S. maltophilia showed higher adhesion compared to bacteria grown in LB broth, to both biotic and abiotic surfaces. However, when bacteria were grown in LB broth supplemented with different concentrations of glucose, under aerobic conditions, the bacteria grown at lower glucose concentration (2 gm/l) showed maximum adhesion to abiotic surfaces (polystyrene microtiter plate) compared to biotic surfaces (mouse trachea, mouse tracheal mucus and HEp-2 cells line). Maximum adhesion to biotic surfaces was seen with cells grown at 4 gm/l of glucose concentration. On the contrary if the cell was grown under microaerophilic conditions maximum adhesion to abiotic and biotic surfaces was achieved with bacteria grown at 1 gm/l and 2 gm/l of glucose concentration respectively. A negative correlation was observed between glucose concentrations and pH of media, the latter declined faster under microaerophilic conditions as compared to aerobic condition.     

Heat-induced expression and chemically induced expression of the Escherichia coli stress protein HtpG are affected by the growth environment.

Differences in expression of the Escherichia coli stress protein HtpG were found following exposure of exponentially growing cells to heat or chemical shock when cells were grown under different environmental conditions. With an htpG::lacZ reporter system, htpG expression increased in cells grown in a complex medium (Luria-Bertani [LB] broth) following a temperature shock at 45 degrees C. In contrast, no HtpG overexpression was detected in cells grown in a glucose minimal medium, despite a decrease in the growth rate. Similarly, in pyruvate-grown cells there was no heat shock induction of HtpG expression, eliminating the possibility that repression of HtpG in glucose-grown E. coli was due to catabolite repression. When 5 mM phenol was used as a chemical stress agent for cells growing in LB broth, expression of HtpG increased. However, when LB-grown cells were subjected to stress with 10 mM phenol and when both 5 and 10 mM phenol were added to glucose-grown cultures, repression of htpG expression was observed. 2-Chlorophenol stress resulted in overexpression of HtpG when cells were grown in complex medium but repression of HtpG synthesis when cells were grown in glucose. No induction of htpG expression was seen with 2, 4-dichlorophenol in cells grown with either complex medium or glucose. The results suggest that, when a large pool of amino acids and proteins is available, as in complex medium, a much stronger stress response is observed. In contrast, when cells are grown in a simple glucose mineral medium, htpG expression either is unaffected or is even repressed by imposition of a stress condition. The results demonstrate the importance of considering differences in growth environment in order to better understand the nature of the response to an imposed stress condition.     

Adhering ability of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> is dependent on growth conditions

The growth conditions are known to influence the bacterial adhesion to different kinds of surfaces. In the present study the adhering ability of S. maltophilia, on growth in nutrient rich media (Tryptic Soy Broth (TSB)) and minimal media (Luria Bertani (LB)) was checked by viable cell count and spectrophotometric method. TSB grown S. maltophilia showed higher adhesion compared to bacteria grown in LB broth, to both biotic and abiotic surfaces. However, when bacteria were grown in LB broth supplemented with different concentrations of glucose, under aerobic conditions, the bacteria grown at lower glucose concentration (2 gm/l) showed maximumadhesion to abiotic surfaces (polystyrene microliter plate) compared to biotic surfaces (mouse trachea, mouse tracheal mucus and HEp-2 cells line). Maximum adhesion to biotic surfaces was seen with cells grown at 4 gm/l of glucose concentration. On the contrary if the cell was grown under microaerophilic conditions maximum adhesion to abiotic and biotic surfaces was achieved with bacteria grown at 1 gm/l and 2 gm/l of glucose concentration respectively. A negative correlation was observed between glucose concentrations and pH of media, the latter declined faster under microaerophilic conditions as compared to aerobic condition.     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

Effects of growth medium selection on plasmid DNA production and initial processing steps

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Genetic evidence of distinct physiological regulation mechanisms in the sigma(54) Pu promoter of Pseudomonas putida

The activity of the toluene-responsive sigma(54) Pu promoter of the pWW0 TOL plasmid of Pseudomonas putida is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. Although the Pu promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of beta-galactosidase in a Pu-lacZ reporter P. putida strain. Since Pu was still down-regulated during exponential growth regardless of glucose addition, it appeared that the carbohydrate separately influenced promoter activity. This notion was supported by the growth-dependent induction pattern of Pu in a ptsN mutant of P. putida, the loss of which makes Pu no longer responsive to repression by glucose. On the other hand, overexpression of the sigma factor sigma(54), known to partially alleviate the exponential silencing of the promoter, did not affect glucose inhibition of Pu. These data indicated that exponential silencing and carbon source-dependent repression are two overlapping but genetically distinguishable mechanisms that adapt Pu to the physiological status of the cells and nutrient availability.     

The Escherichia coli CsrB and CsrC small RNAs are strongly induced during growth in nutrient-poor medium

The carbon storage regulatory (Csr) system is a complex network controlling various phenotypes in many eubacteria. So far, the external conditions by which the system is regulated are poorly understood. Here we show that the expression of the two noncoding small RNAs CsrB and CsrC in Escherichia coli is strongly increased in cultures grown in minimal medium. Addition of tryptone, casamino acids or a mixture of amino acids to a culture grown in minimal medium led to a rapid reduction in the levels of CsrB. Based on this we propose that the expression of the Csr sRNAs is controlled by the amino acid availability in the growth medium.     

Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny

AIMS: To assess the abilities of 105 avian pathogenic Escherichia coli (APEC) and 103 avian faecal commensal E. coli (AFEC) to form biofilms on a plastic surface and to investigate the possible association of biofilm formation with the phylotype of these isolates. METHODS AND RESULTS: Biofilm production was assessed in 96-well microtitre plates using three different media, namely, M63 minimal medium supplemented with glucose and casamino acids, brain-heart infusion broth, and diluted tryptic soy broth. Avian E. coli are highly variable in their ability to form biofilms. In fact, no strain produced a strong biofilm in all three types of media; however, most (75.7% AFEC and 55.2% APEC) were able to form a moderate or strong biofilm in at least one medium. Biofilm formation in APEC seems to be mostly limited to nutrient deplete media; whereas, AFEC are able to form biofilms in both nutrient deplete and replete media. Also, biofilm formation in E. coli from phylogenetic groups B2, D and B1 was induced by nutrient deplete conditions; whereas, biofilm formation by members of phylogenetic group A was strongest in a rich medium. CONCLUSIONS: Biofilm formation by APEC and phylotypes B2, D and B1 is induced by nutrient deplete conditions, while AFEC are able to form biofilms in both nutrient rich and deplete media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate biofilm formation by a large sample of avian E. coli isolates, and it provides insight into the conditions that induce biofilm formation in relation to the source (APEC or AFEC) and phylogenetic group (A, B1, B2 and D) of an isolate.     

Cell volume increase in Escherichia coli after shifts to richer media.

Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth. Cell numbers and mean cell volumes were measured electronically. The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient. Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures. When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division. When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values. In all cases, the cell volume increase was linear both before and after the growth rate transition. The strongest support for a linear cell volume increase during the cell cycle of E. coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates. Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems.     

Thiamine triphosphate, a new signal required for optimal growth of Escherichia coli during amino acid starvation

Thiamine triphosphate (ThTP) is present in low amounts in most organisms from bacteria to humans, but its biological role remains unknown. Escherichia coli grown aerobically in LB medium contain no detectable amounts of ThTP, but when they are transferred to M9 minimal medium with a substrate such as glucose or pyruvate, there is a rapid but transient accumulation of relatively high amounts of ThTP (about 20% of total thiamine). If a mixture of amino acids is present in addition to glucose, ThTP accumulation is impaired, suggesting that the latter may occur in response to amino acid starvation. To test the importance of ThTP for bacterial growth, we used an E. coli strain overexpressing a specific human recombinant thiamine triphosphatase as a glutathione S-transferase (GST) fusion protein (GST-ThTPase). Those bacteria were unable to accumulate measurable amounts of ThTP. On minimal medium supplemented with glucose, pyruvate, or acetate, they exhibited an intermediate plateau in cell growth compared with control bacteria expressing GST alone or a GST fusion protein unrelated to thiamine metabolism. These results suggest that the early accumulation of ThTP initiates a reaction cascade involved in the adaptation of bacteria to stringent conditions such as amino acid starvation. This is the first demonstration of a physiological role of this ubiquitous compound in any organism.     

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.     

Constancy of growth on simple and complex media.

An apparatus has been developed in which bacterial growth can be measured very precisely over short intervals of time. Its precision is presented and used to assess the constancy of growth in batch culture. Under certain conditions, i.e., Luria broth or 0.2% glucose-M9 medium at very low cell densities, the specific growth rate of Escherichia coli appeared to be constant within the measurement limits of the method. In succinate minimal medium, the growth rate increased gradually over several days and never became constant. With nutrient broth and with Luria broth, growth slowed progressively at moderate cell densities within the range considered to be in the logarithmic phase of growth. In addition, temporary slowdown in growth rate occurred in these two complex media at characteristic cell densities. These gradual increases in succinate minimal medium and temporary slowdowns in the complex media would be undetectable without precise measurements and may have been a source of variability in many bacteriological studies.     

Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.