The results of our experiments have shown that DNA microarray analysis is a valuable tool for analysis of global gene expression in response to environmental stimuli. Whereas E. coli
K-12 arrays have been used in a number of investigations (13
), the E. coli
O157 array opens the door for transcriptome analysis of this serious bacterial pathogen.
Little is known about the global genetic regulation of E. coli
O157:H7 genes. Sperandio et al. (45
) have shown by DNA microarray analysis that quorum sensing is a global regulatory mechanism for basic physiological functions as well as for the production of virulence factors in E. coli
O157:H7. They found that 404 genes were upregulated by luxS
at least fivefold. Moreover, an involvement of the SOS response was indicated in their study. Whereas the transcription of stx2
was 3-fold higher in strain 86-24 than in a luxS
mutant, SOS genes such as recA
, and sulA
were upregulated more than 20-fold. Dahan et al. (8
) investigated the changes in gene expression upon binding of E. coli
O157:H7 to plasma membranes. They found increased levels of stress-associated mRNA and decreased levels of mRNA encoding proteins involved in translation and type III secretion.
Bacteria are able to rapidly respond to environmental signals (24
). Besides signal transduction systems that forward stimuli from the outside of the cell through the cell wall to the inside, chemical substances may enter the cells and directly or indirectly affect gene transcription. Bacterial pathogens interact with a specific environment, the human host. Besides interaction with epithelial surfaces and competitive growth with the commensal flora, they come into contact with chemicals that are used for therapy. One group of chemicals used most frequently for the treatment of infectious diseases are antibiotics. Antibiotics are thought to interact specifically with bacterial metabolism and target specific structures. Targeting of these structures with therapeutic concentrations of antibacterial chemicals inhibits bacterial growth by interfering with cell wall biosynthesis, nucleotide synthesis, or protein biosynthesis. Previous studies from a number of investigators have shown that the mechanisms of antibiotic action are more complex and that subinhibitory concentrations of antibiotics may alter the bacterium's properties (2
). Since the use of antibiotics is common in veterinary and human medicine, as well as in animal husbandry and the food industry worldwide, bacteria frequently come into contact with antibiotics. Certain antibiotics may cause induction of bacteriophages, i.e., Stx-encoding bacteriophages (21
), and even subinhibitory concentrations may modulate Stx production (11
Norfloxacin is a member of the group of 4-quinolone antibiotics that interact with both DNA gyrase and topoisomerase IV (10
). For E. coli
, DNA gyrase is the primary target. Quinolones inhibit DNA synthesis by triggering the formation of stable complexes of DNA and topoisomerase IV as well as DNA gyrase. The quinolone-mediated cell death is thought to be initiated by the generation of double-stranded breaks (10
). Norfloxacin is a good inducer of the SOS response, and it has been shown that genes of the SOS regulon are induced on E. coli
K-12 microarrays upon incubation with norfloxacin in a dose-dependent manner (44
). Those investigators used concentrations ranging from 0.03 to 8.0 μg of norfloxacin per ml for 30 min. The highest extent of gene regulation was observed with the highest norfloxacin concentration, which caused the upregulation of 22 genes involved in DNA metabolism. With a norfloxacin concentration comparable to that which we have used, only four genes of the SOS response were upregulated after 30 min of induction.
We have used 200 ng of norfloxacin per ml over an induction period of 120 min and detected by array analysis only two upregulated genes that were associated with the SOS response. One of these encodes a protein homologous to DinI of Serratia marcescens
, and the other one encodes a DinD homologue (Table ) (53
). The ratios for both genes were 2.7 and 5.8, respectively, indicating moderate induction (Table ). The real-time RT-PCR performed for determination of the expression of recA
confirmed the array data for uvrD
and indicated a moderately increased level of recA
expression. In general, RT-PCR is more sensitive because RT can be performed without incorporation of cyanine dyes. It is not clear why only three SOS-associated genes are switched on, but this may depend on the fact that particular DNA-damaging agents can preferentially induce particular genes of the SOS regulon (3
), on the prolonged induction time, or on the low norfloxacin concentration.
The physiological response to norfloxacin probably cannot be compared between E. coli
K-12 and EDL933, since EDL933 probably does not respond with a similar strength as E. coli
K-12. However, 200 ng of norfloxacin per ml induced phage genes; and the genes most strongly affected were those of BP-933W in the late region, including stx2
, which was induced more than 150-fold. It is intriguing that three SOS-related genes were induced. However, a low concentration of norfloxacin could probably switch on alternate DNA repair pathways. E. coli
possesses multiple inducible DNA repair pathways; among these are recA
-independent pathways, such as the UVM response (1
). However, the most plausible explanation would be that the low norfloxacin concentration is not able to fully induce the SOS regulon genes.
Regulation of gene transcription is an important response to environmental changes and is stringently regulated in bacteria. Our present understanding indicates that the coordination of gene transcription in E. coli
involves a number of hierarchical levels, such as the local control of individual operons, the regional control of multiple operons within a regulon, or the regional control of multiple regulons in regulatory networks that are termed stimulons or modulons (29
A high hierarchical level of gene regulation is represented by changes in the DNA conformation, such as DNA supercoiling or the presence of intrinsically curved DNA (14
). In studies with mutants it has been shown that the maximal abundance of proteins in a set of 88 proteins investigated occurred at supercoiling levels below that of the wild type, and others were most abundant with elevated levels of negative superhelices (46
). Since the superhelical status is caused and influenced by the activities of DNA gyrase and topoisomerase, DNA gyrase inhibitors also influence supercoiling and gene transcription (10
). It has been demonstrated that DNA gyrase inhibitors also lower the levels of DNA supercoiling. It may be hypothesized that norfloxacin at higher concentrations inhibits bacterial growth and probably modulates phage gene expression by causing changes in the DNA tertiary structure at lower levels. However, genes that have already been shown by other investigators to be regulated by supercoiling were not upregulated in our study. Future experiments will be needed to investigate this phenomenon.
It is conspicuous that the genes of prophage BP-933W are transcribed most strongly. In concordance with a temporal transcription scheme (48
), genes of the late-phase region are upregulated manyfold (Table ). However, some genes, such as int
, and some ORFs, do not fit in that scheme (Fig. ). In order to obtain reproducible results, we used a strict system for the inclusion of spots in our analyses, as described above. For example, when the ratio for a spot on one of the three experimental slides was less than 2.0, the spot was precluded from further analyses. For the int
gene, we obtained values of 4.0, 5.0, and 1.16 (data not shown). Although int
appeared to be upregulated on two of the slides, it had to excluded since the ratio for one spot was less than 2.0. Therefore, from the slide data we cannot suggest exactly whether this gene is regulated or not. A similar situation was found for some other genes, such as c
I. The bor
gene is transcribed in the opposite direction and is not thought to be cotranscribed with the late phage genes, and lom
was not present in our isolate in a fully intact form and therefore had to be excluded from the analysis.
Another interesting question to be addressed is the high level of expression of stx2
and the difference between the transcription efficiencies of stxA2
. Up to now it has not been clear whether the relation of the A and B subunits of 1:5 is regulated on the transcriptional level or the translational level (30
). By taking into account the fact that slide ratios cannot be considered absolute values, we have validated these data with real-time RT-PCR experiments. The A-subunit genes are transcribed more efficiently than the B-subunit genes. This was not expected and may be interpreted as evidence for regulation at the posttranscriptional level. This suggestion needs further research.
A number of phage genes are demonstratively regulated very strongly. These are mainly BP-933W genes. Whereas the stx2 gene is upregulated more than 150-fold, most genes of the late region displayed ratios from 10- to 99-fold. This is in contrast to CP-933V and non-Stx phages (Tables to ), which show lower regulation ratios, with a maximum of 20-fold. This points to the question of whether the different prophages possess different mechanisms of regulation of their growth cycle.
In older studies it was observed that toxin production is affected by the presence of a second toxin-encoding phage. The same could be true in the context of our study. Repressors of the BP-933W phage may alter gene transcription in CP-933V or the other prophages. Alternatively, these phages could contain other mechanisms of regulation. An interesting experiment would be investigation of whether the DNA supercoiling status of BP-933W may be responsible for that phenomenon.
Microarray analysis opens new dimensions in E. coli O157 research. The fact, however, that E. coli O157:H7 contains so many homologous DNA sequences aggravates the handling of E. coli O157:H7 arrays in this specific field of phage research. Prophage genes, present as functional or defective phage fragments, show gene transcription activity upon induction by environmental stimuli. Future research will be needed to clarify the impact of these observations for population biology and to determine regulation networks that enable the prophages to respond to and communicate with each other and alter transcription activity.