Detection and analysis by dual‐laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli

Abstract

Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual-laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A-T-and G-C-rich regions of DNA, respectively. An exponentially-growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:l multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanolfixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty-five minutes post-infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA-containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4DNA inside E. coli. A value of 35.6 k 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that duallaser flow cytometry can be used to study viral DNA inside the bacterial host.Key terms: Hoechst 33258, chromomycin A3, DNA base composition, viral DNA, determination of % G + C The combination of two DNA-specific f luorochromes, Hoechst 33258 (H33258) and chromomycin A3 (CA3), has been used to study mammalian chromosomes and bacterial DNA (5, 6, 9, 10,17,22). H33258 binds preferentially to regions of DNA rich in A-T base pairs (3, 14) while CA3 binds preferentially to G-C-rich regions of DNA (2). The ratio of CA3:H33258 relative fluorescence measured by dual-laser flow cytometry has been shown to be related to the base composition of bacterial DNA (22) and has been used to determine the % G + C of DNA in bacteria (17).Using H33258 and CA3 in combination, it may be possible to detect bacteriophage DNA inside of its bacterial host and to determine the % G + C of the bacteriophage DNA using the CA3:H33258 fluorescence ratio. It has been shown that the combination of H33258 and CA3 can be used to differentiate bacterial populations in mixtures containing two species of bacteria which differ in DNA base composition by as little as 4% G + C (17). The DNA of bacteriophage T4 has a % G + C of 34-36.1 (12, 15, 18, 21) which differs in base composition from the DNA of its host, E . coli 148-52% G + C (1611, by greater than 4% G + C. Because of the wide difference in the % G + C, it should be possible, in theory, to distinguish T4 DNA from E. coli DNA using CA3 versus H33258 fluorescence.Flow cytometric analysis of T4 DNA inside E. coli cells is somewhat more complex than analysis of bacterial DNA by flow cytometry. The presence of both T4 DNA and host cell DNA within E. coli cells contribute to the fluorescence associated with CA3-and H33258-stained E . coli cells infected with T4. However, relatively early in the course of infection of E . coli by T4, the chromosome of the host is degraded by viral nucleases (13). As degradation of host cell ...

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

“…In the past decade, flow cytometry has been used for measuring cell cycle-related properties of a variety of mammalian cell cultures and has had major impact on cell cycle studies of mammalian cells. Recently, flow cytometry techniques have been successfully employed in cell cycle studies in the yeast Saccharomyces cerevisiae (11,12,43) and in DNA replication studies in the bacterium E. coli (6,38,41,(44)(45)(46)(47)52).…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…Diatoms (Bacillariophyeeae) Phaeodactylum tricornotum [48] Dinoflagellates Gonyaulax polyedra [ 197] Viruses Eseherichia coli bacteriophage T4 [168,182] Pox virus [182] a Only the species which were not cited in our previous review [76] are mentioned.…”

“…Analysis of small and large microorganisms can be performed by most existing instruments, including those devoted to analysis with air-cooled low-power laser [173] or mercury arc lamp illumination [182,183], provided the precise conditions described above are respected. Although very small bacteria are at the limit of detection of commercial flow cytometers, some instruments may even detect large viruses when used by skilled operators [168,182].…”

“…It should be kept in mind that these fluorochromes have a higher specificity for AT base pairs and may not reflect the total DNA content. The pioneering work of van Dilla et al has been pursued [192] and the AT/GC contents of flagellates [128,140], various bacterial species [167], and bacteriophage T4 [168] have been determined using the same combination of fluorochromes (Hoechst 33258 and chromomycin A3) as those used for mammalian chromosome discrimination [122]. With air-cooled He-Cd lasers, the combination of DAPI with olivomycin successfully differentiated S aureus, E coli and P aeruginosa [173].…”

“…Recent technical breakthroughs. Yeast and bacteria labelling by hybridization in suspension with RNA probes [7, 22, 215] Detection of DIG reporter molecule inside Gram-negative bacterial cells by antibodies [215] -lmmunoanalysis of bacterial outer membrane proteins [15, 101, 130, 164, 181] -Detection of internal antigen in yeast or in slime mould cells [21, 57, 64, 65] -Yeast cell cycle analysis by BrdU/DNA double staining method [57]Growth and enzymatic activity determination of bacterial and yeast cells entrapped in microbeads[135][136][137]200] -Slit-scan of yeast cells[24] Analysis of GC content of protozoa, bacteria and bacteriophages[41,128,140,167,168] -Vacuolar pH measurement in yeast…”