Characterization of the ftsZ gene from Ehrlichia chaffeensis,Anaplasma phagocytophilum,and Rickettsia rickettsii,and use as a differential PCR target

Degenerate primers corresponding to highly conserved regions of previously characterized ftsZ genes were used to PCR amplify a portion of the ftsZ gene from the genomic DNA of Ehrlichia chaffeensis (ftsZ(Ech)), Anaplasma phagocytophilum (ftsZ(Ap)), and Rickettsia rickettsii (ftsZ(Rr)). Genome walking was then used to amplify the 5' and 3' termini of the genes. The DNA sequences of the resulting amplification products yielded open reading frames coding for proteins with molecular masses of 42.0, 45.7, and 48.3 kDa for A. phagocytophilum, E. chaffeensis, and R. rickettsii, respectively. These homologs are 20 to 70 amino acids longer than the FtsZ proteins characterized in bacteria such as Escherichia coli and Bacillus subtilis, but do not possess the large extended carboxyl-termini found in the FtsZ proteins of Bartonella, Rhizobium, and Agrobacterium species. The functional domains important for FtsZ activity are conserved within the ehrlichial and rickettsial FtsZ protein sequences. The R. rickettsii FtsZ sequence is highly homologous to the FtsZ protein previously described for Rickettsia prowazekii (89% identity), and identical to the FtsZ protein of Rickettsia conorii. The percent identity observed between the A. phagocytophilum and E. chaffeensis FtsZ proteins is only 79% and is particularly low in the carboxyl-terminal region (15.8% identity). Primers were designed to PCR amplify a portion of the variable carboxyl-terminal region of the ftsZ gene, and used to differentiate each agent based on the size of the amplicons: A. phagocytophilum, 278 bp; E. chaffeensis, 341 bp; and Rickettsia spp., 425 bp.     

Borrelia burgdorferi ftsZ plays a role in cell division

ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZ(Bbu)). Its gene product (FtsZ(Bbu)) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZ(Bbu) might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZ(Bbu) antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZ(Bbu) could interfere with the function of E. coli FtsZ, ftsZ(Bbu) was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZ(Bbu) generated a filamentous phenotype. This suggested interference of ftsZ(Bbu) with E. coli FtsZ function and confirmed the role of ftsZ(Bbu) in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.     

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall.

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.     

Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity.

Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation. Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B. subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ. Previously, we cloned and sequenced the B. subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species. We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds. Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes. It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated. The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation. Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C. Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant. The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues).     

Reconstitution of nucleotide excision nuclease with UvrA and UvrB proteins from Escherichia coli and UvrC protein from Bacillus subtilis

Recently, an open reading frame which has a deduced amino acid sequence that shows 38% homology to Escherichia coli UvrC protein was found upstream of the aspartokinase II gene (ask) in Bacillus subtilis (Chen, N.-Y., Zhang, J.-J., and Paulus, H. (1989) J. Gen. Microbiol. 135, 2931-2940). We found that plasmids containing this open reading frame complement the uvrC mutations in E. coli. We joined the open reading frame to a tac promoter to amplify the gene product in E. coli and purified the protein to near homogeneity. The apparent molecular weight of the gene product is 69,000, which is consistent with the calculated molecular weight of 69,378 fro the deduced gene product of the open reading frame. The purified gene product causes the nicking of DNA at the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to a thymine dimer when mixed with E. coli UvrA and UvrB proteins and a DNA substrate containing a uniquely located thymine dimer. We conclude that the gene product of the open reading frame is the B. subtilis UvrC protein. Our results suggest that the B. subtilis nucleotide excision repair system is quite similar to that of E. coli. Furthermore, complementation of the UvrA and UvrB proteins from a Gram-negative bacterium with the UvrC protein of Gram-positive B. subtilis indicates a significant evolutionary conservation of the nucleotide excision repair system.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Cloning and expression of the gene for the mycoplasma key division protein FtsZ in Escherichia coli]

Gene ftsZ responsible for division of bacterial cells was revealed in most prokaryote groups. A 520-bp fragment of the ftsZ gene was amplified on the template of A. laidlawii DNA using degenerate primers. This fragment was sequenced and served as a hybridization probe for cloning of the full-sized copy of the A. laidlawii ftsZ gene. The amplified fragment was cloned in a pGEX3X vector and expressed in E. coli cells. Polyclonal antibodies derived from the chimeric polypeptide containing a fragment of A. laidlawii FtsZ protein interacted only with the A. laidlawii protein with molecular mass of 40 kDa. Comparison of nucleotide sequences of the ftsZ-gene region of A. laidlawii and other bacterial species showed that they were highly homologous in A. laidlawii, E. coli, and Bac. subtilis, while low homology was revealed between the A. laidlawii sequence and those of the members of the genus Mycoplasma. Analysis of the ftsZ-gene nucleotide sequences is suggested as a means to study the evolutionary relatedness of prokaryotes.