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The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the -10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the -10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species., (Copyright © 2020 American Society for Microbiology.)

To identify interactions of chromatin proteins with the genome of the cell type of interest that is a part of heterologous tissues and organs of Drosophila, an FLP-dependent DamID approach was recently developed [4], which does not require sorting of cells or nuclei. Here, a modification of this approach, Dam inv , is described. The modified approach was validated by generating the binding pattern of the LAM protein, a component of the inner membrane of the nuclear envelope, with the genome of glial cells of the Drosophila larval central brains.

Conjugative plasmids and conjugative transposons contain the genes, which products specifically inhibit the type I restriction--modification systems. Here is shown that non-conjugative transposons Tn2l, Tn5053, Tn5045, Tn501, Tn402 partially inhibit the restriction activity of the type I restriction-modification endonuclease EcoKI (R2M2S1) in Escherichia coli cells K12 (the phenomenon of restriction alleviation, RA). Antirestriction activity of the transposons is determined by the MerR and ArdD proteins. Antirestriction activity of transposons is absent in mutants E. coli K12 clpX and clpP and is decreased in mutants E. coli K12 recA, recBC and dnaQ (mutD). Induction of the RA in response to the MerR and ArdD activities is consistent with the production of unmodified target sequences following DNA repair and DNA synthesis associated with recombination repair or replication errors. RA effect is determined by the ClpXP-dependent degradation of the endonuclease activity R subunit of EcoKI (R2M2S1).

DNA methylation catalyzed by DNA methyltransferases (MTases) is widespread in prokaryotes. In an attempt to find EcoDam variants with enhanced preference for hemimethylated DNA, the L122, P134, and V133 residues were replaced with other amino acids using site directed mutagenesis, and the catalytic activity of all variants on unmethylated and hemimethylated substrates was studied. Our results showed that, in addition to L122A, the L122S and L122A/V133L EcoDam variants were able to sense the methylation status of the 5'-GATC-3' double-stranded target recognition site and methylated only hemimethylated DNA.

When transformed into Escherichia coli K12 strains, the mercury resistance transposon Tn5053@ exhibits high antirestriction activity against the EcoKI type I restriction and modification system. The products of the genes merR and ardD contribute to the antirestriction activity of Tn5053. The merR gene encodes the MerR protein, the transcription regulator of the mer operon genes. The ardD gene is responsible for ArdD protein synthesis and is located within the tni operon. In the following study, it was demonstrated that the antirestriction activity of the transposon Tn5053 is absent in E. coli K12 strains with the mutant genes clpX, clpP, and recA. The antirestriction effect of Tn5053 is not enhanced by 2-aminopurine. The Tn5053 antirestriction activity is not altered in E. coli K12 with the mutant dam gene; however, it is decreased in the E. coli K12 mutD. It is assumed that the activities of the MerR and ArdD proteins lead to the formation of a significant amount of unmodified DNA in the bacterial cell, causing the SOS-dependent reduction of the EcoKI (R2M2S) enzyme activity associated with ClpXP-induced proteolysis of the R-subuinit.

The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3' end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.

Rpn4p is a transcription factor responsible for coordinated regulation of proteasomal genes in Saccharomyces cerevisiae. This factor is involved directly or indirectly in regulation of comprise more than one tenth part of all yeast genome. Traditional methods are inappropriate for mapping of Rpn4p binding sites because of its extremely low concentration in the cell. We have developed the model system using Dam-methylase of E. coli which allows to detect interaction of Rpn4p with its target genes. In this system we showed that Rpn4p is recruited to proteasomal genes only through interactions with DNA.

The ArdA and Ocr antirestriction proteins, whose genes are in transmissible plasmids (ardA) and bacteriophage genomes (0.3 (ocr)), specifically inhibit type I restriction-modification enzymes. The Ocr protein (T7 bacteriophage) was shown to inhibit both restriction (endonuclease) and modification (methylase) activities of the EcoKI enzyme in a broad range of intracellular concentrations (starting from 10-20 molecules per cell). In contrast to Ocr, the ArdA protein (ColIb-P9 transmissible plasmid) inhibited both of the EcoKI activities only at high intracellular concentrations (30000-40000 molecules per cell). When the ArdA concentration was several fold lower, only endonuclease activity of EcoKI was inhibited. It was assumed that a poorer ArdA ability to inhibit EcoKI modification activity is related to the substantial difference in life cycle between transmissible plasmids (symbiosis with the bacterial cell) and bacteriophages (infection and lysis of bacteria). The Ocr and ArdA mutants that inhibited exclusively endonuclease activity of EcoKI were obtained. Antirestriction proteins incapable of homodimerization were assumed to inhibit only endonuclease activity of type I restriction-modification enzymes.

We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

The EcoDam and T4Dam DNA-(adenine N6)-methyltransferases both methylate the adenine residue in GATC sites. These enzymes are highly related in amino acid sequence, but they deviate in their contact to the first base pair of the target sequence. EcoDam contacts Gua1 with K9 (which corresponds to T4Dam A6), while T4Dam contacts Gua1 with R130 (which corresponds to EcoDam Y138). We have "transplanted" the T4Dam DNA recognition into EcoDam and show that the EcoDam K9A/Y138R double mutant is highly active and specific. We also studied the intermediates of this transition: The EcoDam K9A variant showed low activity and loss of recognition of Gua1 [Horton, et al., J. Mol. Biol. 2006, 358, 559-570]. In contrast, the EcoDam Y138R variant, which carries both Gua1 recognition elements (K9 from EcoDam and R138 corresponding to R130 from T4Dam), is fully active and specific. This result indicates that a smooth evolutionary pathway exists for changing the EcoDam DNA recognition mode to T4Dam without loss of activity and without generation of evolutionary intermediates with reduced activity. We consistently observed increased activity of EcoDam variants containing Y138R; this suggests that the transition from EcoDam (Gua1 recognition through K9) to T4Dam (Gua1 recognition through R130) was driven by selective pressure towards increased catalytic activity.

We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg(95), Asn(126), Asn(132), Arg(116), and Lys(139), which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg(137), which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact k(cat)/K(m)(DNA) or DNA affinity (K(D)(DNA)). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant k(chem)/K(D)(DNA). These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (k(cat)/K(m)(DNA)) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg(95), Asn(126), Asn(132), and Arg(116)) repress the modulation of the response of the enzyme to flanking sequence effects. Processivity impacted mutants do not show substrate-induced dimerization as is observed for the wild type enzyme. This study describes the structural means by which an enzyme that does not completely enclose its substrate has evolved to achieve processive catalysis, and how interactions with DNA flanking the recognition site alter this processivity.

Antirestriction protein Ocr (bacteriophage T7) is specific inhibitor of the type I restriction-modification enzymes. The bacteriophage T7 0.3 (ocr) gene is cloned in pUC18 vector. It was shown that T7 Ocr protein inhibits both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The mutation form of Ocr-Ocr F53D A57E, which inhibits only the restriction activity of EcoKI-enzyme, was constructed. The T7 0.3 (ocr) and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltet0-1) promoter which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltet0-1) promoter can be regulated over and up to 5000 fold range by supplying anhydrotetracycline (aTc) to the E. coli MG1655Z1 tetR+ cells. It was determined the dependence of the effectiveness of the antirestriction activity of the Ocr and Ocr F53D A57E proteins on the intracellular concentration. It was shown that the values of the dissociation constants K(d) for Ocr and Ocr F53D A57E proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, K(d) (Ocr F53D A57E) = 10(-7) M.

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the alpha-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.

Aims: The aim of this study was to gain a better understanding of the reason for the predicted pulsed-field gel electrophoresis (PFGE) pattern for the sequenced Escherichia coli O157:H7 EDL933 (EDL933) being different from that observed in practice, using the restriction enzyme Xba1., Methods and Results: Primers were designed that flanked either side of each of the predicted Xba1 restriction sites, and the resultant PCR products were sequenced. No sequencing errors were found in the published genome. The distribution of dam methylation sites within the genome was investigated, and a new PFGE pattern was predicted by assuming that any Xba1 restriction site that coincided with a dam methylation site would not be cut. The estimated mean band sizes were obtained from six replicate gels. It was found that the observed and predicted PFGE patterns were in good agreement., Conclusions: The difference between the observed and the predicted PFGE patterns for EDL933, using Xba1, could be accounted for by assuming that the methylated restriction sites were not cut., Significance and Impact of the Study: PFGE is commonly used as a subtyping method. This study provides additional information about the basic technique that could enhance the interpretation of PFGE patterns in comparative studies of the E. coli isolates.

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.

SeqA and Dam proteins were known to be responsible for regulating the initiation of replication and to affect the expression of many genes and metabolisms. We have examined here the fatty acids composition and phospholipids membrane in dam and/or seqA mutants. The dam mutant showed an accumulation of the acidic phospholipids cardiolipin, whereas, the seqA mutant showed a higher proportion of phosphatidylglycerol compared with the wild-type strain. The seqA dam double mutant showed an intermediate proportion of acidic phospholipids compared with the wild-type strain. Based on these observations, we discuss the role of Dam and SeqA proteins in the regulation of phospholipids synthesis.

Active containment systems based on the controlled expression of a lethal gene are designed to increase containment of recombinant micro-organisms used for environmental applications. A major drawback in containment is the existence of mutations that generate surviving cells that cease to respond to the toxic effect of the lethal function. In this work the authors have developed for the first time a strategy to reduce the problem of mutations and increase the efficiency of containment based on the combination of two lethal functions acting on different cellular targets of major concern in containment, DNA and RNA, and whose expression is under control of different regulatory signals. To engineer the dual gene containment circuit, two toxin-antitoxin pairs, i.e. the colicin E3-immunity E3 and the EcoRI restriction-modification systems, were combined. The genes encoding the immunity E3 and the EcoRI methyltransferase proteins (antitoxins) were stably inserted into the chromosome of the host cell, whereas the broad-host-range lethal genes encoding the colicin E3 RNase and the EcoRI restriction endonuclease (toxins) were flanking the contained trait in a plasmid. This dual lethal cassette decreased gene transfer frequencies, through killing of the recipient cells, by eight orders of magnitude, which provides experimental evidence that the anticipated containment level due to the combination of single containment systems is generally achieved. Survivors that escaped killing were analysed and the mutational events involved were characterized.

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.

Regulated expression of the Escherichia coli dam gene has been achieved with the araBAD promoter lacking a ribosome binding site. Cultures of dam mutants containing plasmid pMQ430 show no detectable methylation in the absence of arabinose and complete methylation in its presence. Dam methyltransferase is a substrate for the Lon protease.

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.

Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a sigma(70)-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn'-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from -16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation

In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 gene is mediated by DNA methylation and the global regulator OxyR. Transcription of agn43 occurs (ON phase) when three Dam target sequences in the agn43 regulatory region are methylated, which prevents the repressor OxyR from binding. Conversely, transcription is repressed (OFF) when these Dam target sequences are unmethylated and OxyR binds. A change in expression phase requires a concomitant change in the DNA methylation state of these Dam target sequences. To gain insight into the process of inheritance of the expression phase and the DNA methylation state, protein-DNA interactions at agn43 were examined. Binding of OxyR at agn43 was sufficient to protect the three GATC sequences contained within its binding site from Dam-dependent methylation in vitro, suggesting that no other factors are required to maintain the unmethylated state and OFF phase. To maintain the methylated state of the ON phase, however, Dam must access the hemimethylated agn43 region after DNA replication, and OxyR binding must not occur. OxyR bound hemimethylated agn43 DNA, but the affinity was severalfold lower than for unmethylated DNA. This presumably contributes to the maintenance of the methylated state but, at the same time, may allow for infrequent OxyR binding and a switch to the OFF phase. Hemimethylated agn43 DNA was also a binding substrate for the sequestration protein SeqA. Thus, SeqA, OxyR and Dam may compete for the same hemimethylated agn43 DNA that is formed after DNA replication in an ON phase cell. In isolates with a mutant seqA allele, agn43 phase variation rates were altered and resulted in a bias to the OFF phase. In part, this can be attributed to the observed decrease in the level of DNA methylation in the seqA mutant.

The Helicobacter pylori hpyIM gene encodes a type II DNA methyltransferase that is highly conserved among strains. To investigate the potential role of M.HpyI methyltransferase activity in controlling gene expression in H. pylori, we analyzed gene transcription profiles in wild-type strain J166 and an isogenic hpyIM mutant strain using gene arrays. This analysis showed that the expression of a majority of genes was unaffected by hpyIM mutation, especially in exponential phase cultures. However, in stationary phase cultures and in cells adherent to AGS gastric epithelial cells in vitro, loss of hpyIM function altered the expression of the stress-responsive dnaK operon. Complementation of the hpyIM mutation using a shuttle plasmid encoding a wild-type copy of the gene re-established the wild-type pattern of dnaK operon expression. These data suggested that hpyIM, encoding a DNA methyltransferase, may have a role in H. pylori physiology that supersedes its original function in a type II restriction-modification system.

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.

I have defined conditions under which RepFIC plasmid DNA can be maintained in a state of lowered helical density. In exponentially growing cultures, the DNA of lowered helical density is present in small amounts but never totally absent, suggesting that it is a normal variant of plasmid maintenance. It is fully methylated at frequent sites by dam-methyltransferase, some not previously recognized, further suggesting that the variant is a precursor of replication. The low-helical density plasmid is present in dam hosts, indicating that methylation is not essential for the change in helical density. The lowered helical density is stabilized in lon hosts, suggesting that Lon-protease may remove both the protein(s) that lower the helical density and the dam-methyltransferase after each round of replication.

Strains of Escherichia coli which lack the dam-encoded adenine methylase are mutators due to a reduction in the efficiency of postreplication mismatch repair. In this study, we show that Dam(-) strains are also defective in very-short-patch repair, the system which corrects T/G mismatches arising from the deamination of 5-methylcytosine. This defect is associated with decreased levels of Vsr, the endonuclease which initiates short-patch repair. We also show that production of the dcm-encoded cytosine methylase is unaffected in Dam(-) strains. Since the dcm and vsr genes are cotranscribed, the regulation of Vsr by Dam is probably posttranscriptional.

We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype., (Copyright 2001 Academic Press.)

Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.

Previous comparative studies revealed close similarity among various groups of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases), indicating their common evolutionary origin. We present evidence for a remarkable similarity between the sequence and predicted structure of HemK (a widespread family of putative proteins encoded in genomes from bacteria to humans) and the catalytic domain of the gamma-subfamily of adenine-specific DNA MTases (N6mA MTases). We predict the structure and function of the putative catalytic domain of HemK proteins and speculate that the target-recognizing function may be conferred by the N-terminal variable region.

A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

The type I restriction-modification system EcoR124I recognizes and binds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3'. The methyltransferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to form the endonuclease. The interaction of the methyltransferase with HsdR has been investigated by surface plasmon resonance, showing that there are two non-equivalent binding sites for HsdR which differ in binding affinity by at least two orders of magnitude. DNA footprinting experiments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not increase the size of the footprint. More extensive in situ footprinting experiments using copper-phenanthroline on the DNA-protein complexes formed by M2S, R1M2S and R2M2S also show no difference in the detailed cleavage pattern, with approximately 18 nucleotides protected on both strands in each complex. Thus the HsdR subunit(s) of the endonuclease stabilise the interaction of the M2S complex with DNA, but do not directly contribute to DNA binding. In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleavage in each case, suggesting that the DNA structure in this region is altered in these complexes.

In Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation. The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E. coli PC2 dnaC2(ts) aligned for initiation of chromosome replication. In both seqA- and dam- cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered. After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA+ dam+ cells than in seqA- or dam- cells, but before the release of oriC from sequestration. Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA. These findings, plus analyses of transcript levels in steady-state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC. Accordingly, a proposal for the sequence of events during the interinitiation interval in E. coli is presented based on the evident coupling of transcription to replication.

Most of the Coxiella burnetii isolates (72 of 80) available in our institute share a 2.1 kb NotI fragment. Sequence analysis revealed two incomplete open reading frames (ORF) for putative genes hemA and bcR coding for glutamyl-tRNA-reductase and bicyclomycin resistance protein, respectively. Upon completing the ORF for the hemA gene by sequencing the adjacent NotI/EcoRI fragment a third ORF for the putative prfA gene was determined coding for release factor 1, an enzyme catalysing the last step in protein biosynthesis.