Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate.

New non-detrimental DNA-binding mutants of the Escherichia coli initiator protein DnaA.

The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.

FNR-mediated oxygen-responsive regulation of the nrdDG operon of Escherichia coli.

Transcription of the nrdDG operon, which encodes the class III nucleotide reductase, which is only active under anaerobic conditions, was strongly induced after a shift to anaerobiosis. The induction was completely dependent on the transcriptional activator FNR and was independent of the ArcA-ArcB two-component response regulator system. The nrdD transcript start site was mapped to a position immediately downstream of two FNR binding sites. Transcription of the other two nucleotide reductase operons, nrdAB and nrdEF, did not respond to oxygen conditions in a wild-type background, but nrdAB expression was increased in the fnr mutant under anaerobic conditions.

Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

Flow cytometric analysis showed that the hns205 and hns206 mutants, lacking the abundant nucleoid-associated protein H-NS, have decreased origin concentration, as well as a low number of origins per cell (ploidy). The most striking observation was that the low ploidy was due to a very short replication time, e.g., at 30 degrees C it was halved compared to that of the hns(+) strain. The decreased origin concentration was not caused by a decreased dnaA gene expression, and the hns206 mutant had normal DnaA protein concentrations. The replication phenotypes of the hns206 mutant were independent of RpoS. Cells overproducing H-NS from a LacI-controlled plasmid had a normal origin concentration, indicating that H-NS is not controlling initiation. A wild-type H-NS concentration is, however, required to obtain a wild-type origin concentration, since cells with an intermediate H-NS concentration had an intermediate origin concentration. Two lines of evidence point to an indirect effect of H-NS on initiation. First, H-NS did not show high-affinity binding to any part of oriC, and H-NS had no effect on transcription entering oriC from the mioC promoter. Second, in a shift experiment with the hns206 mutant, when H-NS protein was induced to wild-type levels within 10 min, it took more than one generation before the origin concentration started to increase.

Characterisation of the allelic variation in the rpoS gene in thirteen K12 and six other non-pathogenic Escherichia coli strains.

The nucleotide sequence of rpoS, the gene for the stress sigma factor, was determined in 13 different K12 strains of Escherichia coli. The results indicate that the original K12 isolate carried an amber mutation at codon 33, which in 50% of the derivatives is mutated by a single base substitution to a coding triplet, in most cases to CAG encoding glutamine. The six non-K12 strains examined here had GAG, encoding glutamate, in position 33. The two most divergent strains had three and seven neutral substitutions in rpoS and carried insertions of 2100 and 2900 bp, respectively, just downstream of the gene. The genetic variations in rpoS were compared with the variation in RpoS-related phenotypes, by measuring catalase (KatE) activity, glycogen accumulation and acid phosphatase levels, and a katEp-gfp fusion was used to visualise katE gene transcription. The RpoS phenotypes of the six rpoS(33E) strains varied significantly more than that of the K12 rpoS(33Q) strains, especially with respect to acid phosphatase levels. This was due to the absence of the gene for the transcriptional activator AppY from four of the rpoS(33E) strains, while all the K12 derivatives carried this gene. When cloned into a LacI-controlled vector and compared in a rpoS::Tn 10 background, the RpoS(33Q) and RpoS(33E) variants showed the same activity.

Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants.

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

Two types of cold sensitivity associated with the A184-->V change in the DnaA protein.

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.

Low-temperature-induced DnaA protein synthesis does not change initiation mass in Escherichia coli K-12.

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.

DnaA boxes are important elements in setting the initiation mass of Escherichia coli.

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.

Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12.

The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold. The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184. In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid. The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells.

H-NS: a modulator of environmentally regulated gene expression.

H-NS is a small chromatin-associated protein found in enterobacteria. H-NS has affinity for all types of nucleic acids but binds preferentially to intrinsically curved DNA. The major role of H-NS is to modulate the expression of a large number of genes, mostly by negatively affecting transcription. Many of the H-NS-modulated genes are regulated by environmental signals, and expression of most of these genes is positively regulated by specific transcription factors. Therefore one of the purposes of H-NS could be to repress expression of some genes under conditions characteristic of a non-intestinal environment, but allow expression of specific genes in response to certain stimuli in the intestinal environment. The hns gene is autoregulated. In vivo the H-NS to DNA ratio is fairly constant except during cold shock, when it increases three- to fourfold. In this review we propose that only the preferential binding to intrinsically curved DNA plays a role under normal growth conditions, and we discuss the different mechanisms by which H-NS might affect gene expression and how H-NS could be involved in the response to different stress situations. Finally, we summarize the evolutionary and functional relationship between H-NS and the homologous StpA.

Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation.

The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.

The histone-like protein H-NS acts as a transcriptional repressor for expression of the anaerobic and growth phase activator AppY of Escherichia coli.

The transcriptional activator AppY is required for anaerobic and stationary-phase induction of the cyx-appA and hya operons of Escherichia coli, and expression of the appY gene itself is induced by these environmental conditions. The sequence of the appY gene and its promoter region is unusually AT rich. The nucleoid-associated protein H-NS has a DNA-binding specificity for intrinsically curved AT-rich DNA. Using a single-copy transcriptional appY-lacZ fusion, we have shown that appY gene expression is derepressed in hns mutants during aerobic exponential growth. In the hns mutant, growth phase and growth rate regulation under aerobic conditions was maintained, while ArcA-dependent anaerobic induction was greatly diminished. Judged by two-dimensional gel electrophoresis, the appY promoter fragment exhibits the features characteristic of curved DNA. Gel retardation assays showed that purified H-NS protein bound with high affinity to two different segments of the appY promoter region. The role of H-NS in the AppY regulatory cascade is discussed and compared with its function in the regulatory cascades of the AppY homologs CfaD and VirF.

Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli.

The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene have been investigated under different environmental conditions with single-copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase. ArcA and AppY activated transcription of the cyx-appA operon during entry into stationary phase and under anaerobic growth conditions. The expression of the cyx-appA operon was affected by the anaerobic energy metabolism. The presence of the electron acceptors nitrate and fumarate repressed the expression of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high level of expression of the operon was obtained in glucose medium supplemented with formate, in which E. coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented in this paper indicate a clear difference in the regulation of the cyx-appA operon and that of the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function under even more-oxygen-limiting conditions than cytochrome d oxidase. The expression of the appY gene is induced immediately by anaerobiosis, and this anaerobic induction is independent of Fnr, and AppY, but dependent on ArcA. The expression of the appY gene is not affected significantly by the anaerobic energy metabolism, i.e., fermentation versus anaerobic respiration. A model incorporating the anaerobic regulation of the appY gene and the two operons which are controlled by AppY, the hydrogenase 1 (hya) operon and the acid phosphatase (cyx-appA) operon, is presented. The expression of the appY gene is inversely correlated with the growth rate and is induced by phosphate starvation as well as during entry into stationary phase. During oxygen-limiting conditions the stationary-phase induction is partially dependent on ArcA. The alternative sigma factor sigma S has limited influence on the transcription of the appY gene during entry into stationary phase and no effect on the induction by phosphate starvation.

The initiation mess?

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the 'initiation mass'. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co-operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective preinitiation DnaA-oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli.

Reversibility of DnaA protein activity in the 'irreversible' dnaA204 mutant of Escherichia coli.

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature down-shift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42 degrees C. The data indicate that the dnaA204 mutant after a shift to 42 degrees C still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30 degrees C, and after induction of DnaA protein synthesis at 42 degrees C. A model describing the processing of DnaA protein in mutants and in the wild type is presented.

Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(nuII) rnh mutant of Escherichia coli.

The kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(nuII) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild-type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild-type concentration (24 ng of DnaA protein per ml cells at OD450 = 1.0). At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30 min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.

Role of the transcriptional activator AppY in regulation of the cyx appA operon of Escherichia coli by anaerobiosis, phosphate starvation, and growth phase.

Transcriptional lacZ fusions have been used to analyze the regulation of the appA operon of Escherichia coli. The appA operon contains the genes cyxA and cyxB, coding for the putative third cytochrome oxidase, and appA, encoding acid phosphatase. The analysis showed that the cyxAB and the appA genes are cotranscribed from a potentially strong promoter, Pcyx, located immediately upstream of cyxA and that the operon in addition contains an internal promoter, PappA, contributing significantly to the transcription of the appA gene. The two promoters were both induced by starvation for Pi and by entry into stationary phase. The cyx promoter was in addition found to be activated by anaerobic growth conditions. The product of the previously identified appY gene, which when present on a high-copy-number plasmid stimulates synthesis of acid phosphatase, was shown to activate the cyx promoter. An insertion mutation in the appY gene was constructed in vitro and recombined into the chromosome. The appY mutation eliminated induction of the cyx promoter by anaerobiosis and severely reduced induction of this promoter by phosphate starvation and upon entry into stationary phase but had no effect on induction of the appA promoter. The appY mutation had no effect on survival in stationary phase, nor did it have any effect on growth rate or yield under aerobic or anaerobic conditions. The possibility that AppY is a third global regulator of energy metabolism genes is discussed.

Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli.

Using a transcriptional fusion to the lacZ gene, we have analyzed the anaerobic regulation of the hydrogenase 1 (hya) operon in response to different anaerobic growth conditions and to mutations in regulatory genes. We found that the transcription of the hya operon was induced when the growth condition was changed from aerobic to anaerobic and that this induction was independent of Fnr but dependent on regulators AppY and ArcA. Furthermore, we found that the transcription of the hya operon was not regulated by the cyclic AMP-cyclic AMP receptor protein complex. Investigation of the effects of different anaerobic growth conditions on the expression of the hya operon showed that expression was induced by formate and repressed by nitrate. Formate induction was not mediated by the fhlA gene product, and nitrate repression was not mediated by the narL gene product. We found a high level of anaerobic expression of the hya operon in glucose medium supplemented with formate and in glycerol medium supplemented with fumarate, suggesting that hydrogenase isoenzyme 1 has a function during both fermentative growth and anaerobic respiration.

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli.

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.