Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution.

We have previously shown that the dnaA gene and the DnaA-box region were conserved in bacteria representative of all three major branches of the eubacterial phylogenic tree: high G + C Gram+, low-G + C Gram+ and Gram-. In the present work, we determined the structure of the dnaA region of Mycoplasma capricolum and found that the dnaA gene and at least two other genes, rpmH and dnaN, were conserved in this bacterium. An unusually high level of amino acid (aa) substitutions was observed in M. capricolum DnaA. It was the case even in those aa which were well conserved in other bacterial species. The nontranslatable region upstream from the dnaA gene was also conserved in this bacterium, as it was universally found in both Gram+ and Gram- bacteria. An additional nontranslatable region downstream from the dnaA gene, which is common to Gram+ bacteria, was also found in M. capricolum, consistent with the proposal that M. capricolum is Gram+ in origin. These regions were rich in A + T and contained ten DnaA-box-like sequences (9-mers that differ from TTATCCACA by one or two bases).     

Genetic structure of the dnaA region of the cyanobacterium Synechocystis sp. strain PCC6803.

We have cloned and sequenced the dnaA region of Synechocystis sp. strain PCC6803, a bacterium with a light-dependent cell cycle. The dnaA gene product, DnaA, is the central factor for replication initiation in bacteria. The deduced amino acid sequence of the protein encoded by the cyanobacterial dnaA gene is 45% identical to DnaA of Bacillus subtilis and fits very well into the homology pattern of the known eubacterial DnaA proteins. The genetic environment of the Synechocystis sp. strain PCC6803 dnaA gene is completely different from the one in other eubacteria. An open reading frame of unknown function, orf134, was detected upstream of dnaA. The purT gene homolog encoding the glycinamide ribonucleotide transformylase T starts about 200 bp away from this open reading frame in the opposite direction. Downstream of the dnaA gene we detected the start of the psbDC operon, which codes for the photosystem II reaction center proteins D2 and CP43 that are involved in the positioning of chlorophyll a.     

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs

Abstract The complete nucleotide sequence of the Borrelia burgdorferi dnaA gene (encoding the initiator protein of chromosome replication) and its flanking regions was determined. The putative DnaA polypeptide exhibited 29–42% identity with those of other eubacteria. The gene order in the dnaA region at the centre of the B. burgdorferi linear chromosome is rnpA-rpmH-dnaN-dnaA-gyrB-gyrA in contrast to the consensus eubacterial order of rnpA-rpmH-dnaN-recF-gyrB , suggesting a rearrangement during the evolution of the Borrelia chromosome. We did not detect the multiple 9-nucleotide repeats known as DnaA boxes, which characterise origin of replications, in the dnaA-gyrB and dnaA-dnaN intergenic regions. In addition B. burgdorferi DnaA protein differs considerably from those of other eubacteria in a normally highly conserved region at the C-terminus of the polypeptide which may be involved in DNA binding.     

Structure and function of DnaA and the DnaA-box in eubacteria: evolutionary relationships of bacterial replication origins

DnaA protein (a trans-acting element) and its binding sequence, DnaA-box: (a cis-acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram-negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA-box (DnaA-box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA-box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA-box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.     

Comparison of dnaA nucleotide sequences of Escherichia coli, Salmonella typhimurium, and Serratia marcescens.

The dnaA genes of Salmonella typhimurium and Serratia marcescens, which complemented the temperature-sensitive dnaA46 mutation of Escherichia coli, were cloned and sequenced. They were very homologous to the dnaA gene of E. coli. The 63 N-terminal amino acids and the 333 C-terminal amino acids of the corresponding DnaA proteins were identical. The region in between, corresponding to 71 amino acids in E. coli, exhibited a number of changes. This variable region coincided with a nonhomologous region found in the comparison of E. coli dnaA and Bacillus subtilis "dnaA" genes. The regions upstream of the genes were also homologous. The ribosome-binding area, one of the promoters, the DnaA protein-binding site, and many GATC sites (Dam methyltransferase-recognition sequence) were conserved in these three enteric bacteria.     

Conserved gene arrangement in the origin region of the Streptomyces coelicolor chromosome.

A 23-kb fragment of the Streptomyces coelicolor chromosome spanning the dnaA region has been isolated as a cosmid clone. Nucleotide sequence analysis of a 5-kb portion shows that the genes for the RNase P protein (rnpA), ribosomal protein L34 (rpmH), the replication initiator protein (dnaA), and the beta subunit of DNA polymerase III (dnaN) are present in the highly conserved gene arrangement found in all eubacterial genomes studied so far. The dnaA-dnaN intergenic region is approximately 1 kb and contains a cluster of at least 12 DnaA boxes with a consensus sequence of TTGTCCACA matching the consensus DnaA box in the phylogenetically related Micrococcus luteus. Two DnaA boxes precede the dnaA sequence. We propose that the chromosomal origin (oriC) of S. coelicolor lies between dnaA and dnaN. In related work, J. Zakrzewska-Czerwinska and H. Schrempf (J. Bacteriol. 174:2688-2693, 1992) have identified the homologous sequence from the closely-related Streptomyces lividans as capable of self-replication.     

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.     

Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

Interactions of the Streptomyces lividans initiator protein DnaA with its target.

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.     

Mycobacterium smegmatis dnaA region and autonomous replication activity.

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.     

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis

Bacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B. subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH. The protein was purified to near homogeneity as judged by SDS-PAGE analysis. The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B. subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences. The purified protein binds ATP with high affinity (Kd = 0.02 microM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus. Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes. The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B. subtilis chromosome showed different levels of growth inhibition when they were introduced into B. subtilis. We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DNaA protein in vitro.