Characterization of genes encoding topoisomerase IV of Mycoplasma genitalium.

A type-II toposiomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involved in cell septation and in the decatenation of newly replicated chromosomes. We have identified parC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium. Since the mechanics of cell septation in conventional eubacterial species is believed to be mediated by cell-wall constituents, there is no clear understanding of what coordinates that process in wall-less species. The presence of par genes in this bacterium, which has the smallest genome of any free-living organism, suggests that Topo-IV has been evolutionarily conserved because of an essential role in mediating cell division.

Origin of replication of the Bacillus subtilis chromosome: in vitro approach to the isolation of early replicating segments.

We have developed a permeable cell system for the study of the molecular mechanisms involved in the control and initiation of DNA replication at the origin of the Bacillus subtilis chromosome. Our system take advantage of the synchronous initiation of DNA replication that occurs in outgrowing B. subtilis spores and the curtailment of DNA elongation by novobiocin. Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nascent DNA chains by affinity chromatography on thiol agarose. The average size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end. The study of the temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB replicated earlier than a segment containing gyrA and part of the rRNA operon (rrnO). This observation is in agreement with previous in vivo data on the replication of origin region and supports the conclusion that the major activity in our in vitro system was the faithful replication of the ori region.

Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation.

We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Genetic map of the Mycoplasma genitalium chromosome.

At 600 kb, the genome of Mycoplasma genitalium is among the smallest known for cellular organisms capable of independent replication. As such, elucidation of the genetic makeup and chromosome architecture of this organism is of considerable interest. We have located 631 markers on the physical map of M. genitalium. The clones have been mapped by hybridizing 20 overlapping cosmid and lambda clones which encompass the entire M. genitalium chromosome to replica filters containing 856 genomic DNA clones. Three hundred fifty-six of these clones represent sequence tag sites, which were previously characterized by database searches. The remaining markers represent clones with an average size of 2.5 kb derived from Sau3A1 partial digestion of genomic DNA. The hybridization data can be divided into three classes: clones which hybridized to only one cosmid; clones which hybridized to two adjacent and overlapping cosmids; and clones which hybridized to several cosmids, which represent repetitive DNA. This rapid approach for placing clones on the physical map has allowed useful comparisons to be made with other bacterial chromosomes, especially that of the closely related organism M. pneumoniae, and has provided insight to the types of events which may have led to the reduction in size of this genome. Future use of these data is discussed.

Construction of an ordered genomic library of Mycoplasma genitalium.

As a first step towards sequencing the chromosome of the suspected human pathogen Mycoplasma genitalium, we attempted to clone its entire genome in a set of ordered cosmids. Cosmid libraries were established by partial digestion of M. genitalium genomic DNA with Sau3AI or EcoRI. A chromosome-walking strategy was used to identify 20 overlapping cosmid clones which contained over 99% of the genome. The final 5.1 kb could not be cloned in cosmids, and was eventually obtained from a genomic library established in a lambda vector. Correspondence of cloned and genomic EcoRI fragments indicated no detectable major deletions or rearrangements in the library. The library was oriented on established XhoI and SmaI physical maps of the chromosome with restriction sites present at the expected locations in the library. The genome contained 74 EcoRI fragments which added up to a total genome size of 578 kb. These were arranged in a partial EcoRI physical map, and those containing the MgPa major attachment protein-encoding operon and its repeat sequences were identified. The existence of this ordered genomic library, which accurately and completely encompasses the entire M. genitalium genome, should serve as a valuable tool for many future studies of this organism and facilitate our long-term goal of sequencing its genome.

An unusual gene containing a dnaJ N-terminal box flanks the putative origin of replication of Mycoplasma genitalium.

Origins of replication are known to be highly conserved among widely divergent microbial species, with the gene order in those regions being dnaA-dnaN-recF-gyrB. On the basis of sequence identities to entries in GenBank, the gene order of a 6-kb fragment of Mycoplasma genitalium DNA was determined to be dnaN-orf311-gyrB-gyrA-serS, which is structurally similar to the ancestral origin of replication. We have directly linked the dnaN gene to the M. genitalium dnaA gene by PCR amplification. However, we found a novel open reading frame, designated orf311, in place of an expected sequence encoding recF. Orf311 contains a DnaJ box motif at its N terminus, but it has no overall homology to any other protein or sequence in the database. We are unable to detect any recF homolog in M. genitalium by hybridization or during a random sequencing survey of the genome.

A survey of the Mycoplasma genitalium genome by using random sequencing.

A total of 508 random clones from five Mycoplasma genitalium genomic libraries were partially sequenced and analyzed. This resulted in the identification of 291 unique contigs. Sequence information from these clones (100,993 nucleotides), representing approximately 17% of this pathogen's genome, was analyzed by comparison to the DNA and protein sequence data bases. The frequency with which clones could be identified, by virtue of possessing homology to another data base entry, was 46%. Sequence analysis indicated the following. (i) The M. genitalium genome contains many genes involved in various metabolic processes. (ii) Repetitive DNA may comprise as much as 4% of this genome. (iii) The MgPa adhesin gene may be the result of horizontal transfer from an unknown origin. (iv) Not all dinucleotide pairs are present in this genome at the expected frequency. (v) This genome potentially encodes approximately 390 proteins and makes very efficient use of its limited amount of DNA. In addition, this study allowed us to estimate the number of genes involved with various cellular functions.

A random sequencing approach for placing markers on the physical map of Mycoplasma genitalium.

A physical map of the Mycoplasma genitalium genome has been prepared using pulsed-field gel electrophoresis. This report details recent efforts made to add markers or specific loci to this map in the absence of any mutants or system of genetic exchange. A total of 44 random clones were partially sequenced. Computer analysis was performed in an attempt to identify homologies with genes already recorded in the DNA sequence database. Clones with a large extent of homology to genes from other microorganisms have been assigned to specific loci on the M. genitalium map by hybridization to selected restriction digests. The additional data has facilitated an updated version of the physical map, and verified this random sequencing method as a useful mapping procedure as well as offering new insight into the physiological processes of this fastidious organism.

A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication.

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis.

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.

Characterization of chromosomal DNA amplifications with associated tetracycline resistance in Bacillus subtilis.

Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tcr) in Bacillus subtilis were first described by C. R. Wilson and A. E. Morgan (J. Bacteriol. 163:445-453, 1985). We have confirmed and extended their results, and we show that fusion of protoplasts from Tcs B. subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 micrograms of tetracycline per ml induces Tcr regenerants that contain amplified DNA. This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol. Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory. All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7. Integration of pCIS7 into the wild-type (Tcs) B. subtilis chromosome and amplification of the plasmid sequences generated a Tcr phenotype, even though the DNA on pCIS7 was cloned from Tcs B. subtilis KS162 (Ives and Bott, J. Bacteriol. 171:1801-1810, 1989). The amplified DNA also showed homology (through hybridization analysis) with pAM alpha 1 delta 1, a gram-positive Tcr plasmid, indicating that B. subtilis normally contains a silent integrated copy of the gene whose amplification confers Tcr. The amplifications were determined to lie between purA and gyrB on the B. subtilis chromosome, and the endpoints were mapped. RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate. The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites. The number of amplified units of DNA was determined through dot blot analysis to do approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.

Mycoplasma pneumoniae DNA gyrase genes.

We have identified a clone from a lambda EMBL3 library containing a 19kb insert of Mycoplasma pneumoniae DNA which includes the genes that encode both subunits of DNA gyrase. The gyrB gene and the 5' end of the gyrA gene have been subcloned into M13. The gyrB gene is 1953bp in length and overlaps the gyrA gene by a single base. The nucleotide sequence of these subclones has significant homology to previously reported gyrase genes. In terms of the size of the gyrB gene and its proximity to the gyrA gene, M. pneumoniae is more similar to Bacillus subtilis than to Escherichia coli.

A physical map of the Mycoplasma genitalium genome.

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse-field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.

Prevalence of novel repeat sequences in and around the P1 operon in the genome of Mycoplasma pneumoniae.

The presence of numerous different repetitive elements in the genome of Mycoplasma pneumoniae has been documented by several laboratories. One which we previously identified, denoted as SDC1, has now been further characterized, verified to be distinct from those discussed in previous publications and shown to lack homology to several other species of Mycoplasma when tested under our stringency conditions. As many as eight versions of the SDC1-type repeat, which is more than 400 bp long, are scattered throughout the genome of M. pneumoniae. The prototype for SDC1 is found within a gene encoding a putative 130-kDa membrane-binding protein lying just downstream from the gene encoding the cytadhesin protein P1. In fact, all of the reported M. pneumoniae repetitive elements have at least one representative either within or adjacent to the P1 operon; many if not all of these lie within open reading frames. The function of these repetitive elements is still unclear.

Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies.

Plasmid pCIS7, containing 11.5 kilobases (kb) of Bacillus subtilis DNA, was isolated from a Tn917 transposon insertion in tetracycline-sensitive B. subtilis KS162. When integrated into the chromosome of B. subtilis 168, this plasmid conferred tetracycline resistance upon reiteration of the plasmid DNA sequences in the chromosome. Deletions and subclones of pCIS7 were constructed and introduced into an Escherichia coli in vitro transcription-translation system. A 72-kilodalton protein was localized to a 3.1-kb PstI-EcoRI fragment of the plasmid. Amplification of the 3.1-kb PstI-EcoRI fragment was required for expression of tetracycline resistance in B. subtilis 168. By hybridization to previously characterized clones, the 11.5-kb fragment was localized to the origin region of the chromosome. Through contour-clamped homogeneous electric field electrophoresis, this cluster of clones was shown to reside on a 200-kb NotI fragment bridging SfiI fragments of 150 and 250 kb and was oriented with respect to the purA and guaA loci, developing an accurate physical map of the region surrounding the origin of replication.

Nucleotide sequence of the P1 attachment-protein gene of Mycoplasma pneumoniae.

The specific attachment of Mycoplasma pneumoniae to the respiratory ciliated epithelium is mediated by a surface protein designated P1. The nucleotide (nt) sequence of the P1 attachment-protein gene has been determined and the amino acid (aa) sequence deduced. mRNA and cDNA sequencing confirm that this gene is transcribed in M. pneumoniae. The predicted amino acid sequence matches the N-terminal 12 aa residues of P1 protein from M. pneumoniae [Jacobs et al., J. Gen. Microbiol. 133 (1987) 2233-2236] beginning with Asn at aa position 60, where aa 1 represents the first codon of the open reading frame (ORF). Notably, the Trp at aa position 69 aligns with a UGA codon deduced from the nucleotide sequence, providing supporting evidence that UGA is read as Trp rather than stop in M. pneumoniae. Analysis of the first 59 aa suggests that it is probably a leader sequence that is processed to yield the mature protein. The codons of the mature P1 protein sequence represent 1568 aa with a calculated Mr of 169,758. A unique feature of this protein sequence is the lack of cysteine, and this was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. pneumoniae proteins metabolically labeled with radioactive cysteine or methionine. This study has revealed that the 4881 nt of the P1 structural gene are flanked by ORFs, and there are no obvious ribosome-binding sites or transcription termination sequences in the immediately adjacent regions. This suggests that the P1 gene is transcribed as part of a larger polycistronic message. In addition, a number of untranscribed and therefore nonfunctional P1 epitope sequences were found in the M. pneumoniae genome; their purpose remains unknown.

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.

Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis.

An 8-kilobase fragment already known to contain the gyrA gene of Bacillus subtilis was shown to encode the gyrB gene as well. Plasmids containing this fragment can rescue both B. subtilis gyrA and gyrB mutants and complement Escherichia coli gyrA mutants. Deletion analysis has indicated the gene locations on the cloned fragment. Under low-stringency conditions the cloned E. coli gyrA and gyrB genes each hybridized to the appropriate subfragments, confirming the assignment of the gene locations on the cloned DNA. In E. coli maxicells, proteins of 67,000 (gyrA) and 77,000 (gyrB) Mr were synthesized. Analysis of proteins encoded by various subfragments indicated the direction of transcription. Although the gyrA and gyrB genes are located adjacent to each other on the chromosome, they may be transcribed independently since expression of gyrA protein is not dependent upon the gyrB gene in maxicells.