Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria

A phylogenetic tree constructed by 5S rRNA analysis is composed of three major branches in eubacteria: high G + C Gram+, low G + C Gram+ and Gram- [Hori and Osawa, Mol. Biol. Evol. 4 (1987) 445-472]. We have shown that the characteristic dnaA region is common among Escherichia coli (Gram-), Pseudomonas putida (Gram-), and Bacillus subtilis (low G + C Gram+). We have now determined the structure of the dnaA region of Micrococcus luteus, as a representative of the last branch, high G + C Gram+. The dnaA gene and at least three other genes, rnpA, rpmH and dnaN were found to be conserved in M. luteus. Large nontranslatable regions were found flanking the dnaA gene. The upstream region is conserved in the four bacteria so far examined. On the other hand, the downstream region is conserved only in Gram+ bacteria, M. luteus and B. subtilis. The consensus sequence of the DnaA box in M. luteus seems to be TTGTCCACA, in contrast to TTATCCACA of other bacteria. These results confirm our hypothesis that the dnaA region is the replication origin of the ancestral bacteria and that the essential feature of the DnaA protein and DnaA-box combination is conserved in eubacteria.     

Comparison of the conserved region in the dnaA gene from three mollicute species

Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria.     

Mapping of replication initiation site in Mycoplasma capricolum genome by two-dimensional gel-electrophoretic analysis.

The homolog of the dnaA gene, which has been reported to be present in the vicinity of the initiation site of replication in the genome of Mycoplasma capricolum (M.Miyata, L.Wang, and T.Fukumura, J. Bacteriol. 175: 655-660, 1993) was mapped precisely. A 9540-bp region containing the dnaA gene was cloned and the entire region was sequenced with the exception of a previously reported region of 2517 bp (Fujita, M.Q., Yoshikawa, H. and Ogasawara, N. Gene 93: 73-78, 1992). The organization of the 9540-bp region was compared with that of corresponding regions in other bacteria. The arrangement and directions of rnpA, rpmH, dnaA, dnaN were conserved, but no other open reading frames were found that were homologous to those that are commonly found around dnaA genes in other bacteria. The directions of movement of the replication fork around the dnaA gene were analyzed by neutral/alkaline two-dimensional gel electrophoresis. The forks developed in a 1569-bp region that consisted of the dnaA structural gene and its downstream non-coding region, and then they proceeded bidirectionally.     

Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes.

To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established with contained the majority of the EcoR1 fragments from M.pneumoniae. The EcoR1 fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome. Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking. We report here the initial results from the sequence analysis of -56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes. The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum.     

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.     

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.     

Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.     

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.     

Genes and their organization in the replication origin region of the bacterial chromosome

Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.     

Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.     

The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.     

Location and sequence analysis of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase-encoding gene (bpdF) of the biphenyl/ polychlorinated biphenyl degradation pathway in Rhodococcus sp. M5

The 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase-encoding gene (bpdF) in the biphenyl (BP)/polychlorinated biphenyl (PCB)-degrading bacterium, Rhodococcus sp. M5 (M5), was found to be located within a 4.5-kb HindIII-BamHI genomic DNA that was 5.4 kb downstream from the bpdC1C2BADE gene cluster. The deduced amino acid (aa) sequence of bpdF revealed that the hydrolase contains 297 aa (32679 Da) that was verified by expression in the Escherichia coli T7 RNA polymerase/promoter system. Unlike previously known HOPD hydrolases, the aa sequence of BpdF appears unique. Interestingly, all HOPD hydrolases and related proteins from the phenol and toluene/ xylene degradation pathways, were found to have a bias in the codon usage in the catalytic Ser within the conserved VGNS(M/F)GG motif.     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA

The nucleotide sequence of a Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCA)-encoding gene (celCCA) and its flanking regions, was determined. An open reading frame (ORF) of 1425 bp was found, encoding a protein of 475 amino acids (aa). This ORF began with an ATG start codon and ended with a TAA ochre stop codon. The N-terminal region of the EGCCA protein resembled a typical signal sequence of a Gram-positive bacterial extracellular protein. A putative signal peptidase cleavage site was determined. EGCCA, without a signal peptide, was found to be composed of more than 35% hydrophobic aa and to have an Mr of 50715. Comparison of the encoded sequence with other known cellulase sequences showed the existence of various kinds of aa sequence homologies. First, a strong homology was found between the C-terminal region of EGCCA, containing a reiterated stretch of 24 aa, and the conserved reiterated region previously found to exist in four Clostridium thermocellum endoglucanases and one xylanase from the same organism. This region was suspected of playing a role in organizing the cellulosome complex. Second, an extensive homology was found between EGCCA and the N-terminal region of the large endoglucanase, EGE, from C. thermocellum, which suggests that they may have a common ancestral gene. Third, a region, which extended for 21 aa residues beginning at aa + 127, was found to be homologous with regions of cellulases belonging to Bacilli, Clostridia and Erwinia chrysanthemi.     

Biological activity of 4-hydroxy-5-formylbenzoic acid derivatives. II. Esters and Schiff bases with antimicrobial activity

A number of hitherto undescribed 4-hydroxy-5-formylbenzoic acid derivatives (A), have been prepared and characterized. (formula; see text) Esters (X = CH3) and Schiff's bases (Z = N-aryl) were prepared by conventional methods and were obtained in satisfactory yield and in a good state of purity. The prepared compounds have been tested for "in vitro" activity against Gram+ bacteria (S.epidermidis, B.subtilis, B.anthracis, M.paratuberculosis), Gram- bacteria (P.aeruginosa, S.typhi murium, E.coli Bb, S.typhi O, S.typhi infantis, S.paratyphi A, S.paratyphi B) and fungi (C.albicans, A.niger, S.cerevisiae) by agar-diffusion method (Kirby-Bauer modified). The prepared compounds, generally, showed inhibitory activity against Gram+ bacteria. Esters (A: X = CH3) showed antibacteric and antimycotic activity. The greatest activity was observed in the methyl ester (XV) of 4-hydroxy-5-formylbenzoic acid (I).