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.

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.

Cloning of the Bacillus subtilis recF gene.

A cloned DNA fragment from the ori region of the Bacillus subtilis chromosome permits three separate recF mutants to grow in the presence of mitomycin C (MC) and survive after ultraviolet (UV) exposure. The recF gene has been localized to a 2.3-kb EcoRI-SalI restriction fragment on the cloned sequences. This fragment directs expression of a 34.7-kDal protein in Escherichia coli maxicells. Cloned DNA containing the recF gene does not complement or rescue recL, recM or spoOJ B. subtilis mutants. The B. subtilis recF gene also does not complement any of several of the E. coli recombination-deficient (Rec-) mutants tested.

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.

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.

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis.

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.

Direct fractionation of genes by preparative electrophoresis of Bacillus subtilis DNA.

Discontinuous electrophoresis through agarose has been shown to be a satisfactory method for preparation of biologically active restriction fragments from milligram quantities of DNA. The DNA is obtained in sufficient quantity for: (1) direct use in genetic transformation, (2) the production of multiple-dimensional restriction analyses, or (3) use as a high-resolution hybridization probe.

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB.

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.

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.

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.

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.

Acrylamide gel electrophoresis of intracellular proteins during early stages of sporulation in Bacillus subtilis.

Acrylamide gel electrophoresis of unfractionated cellular extracts of Bacillus subtilis is shown to be an effective method for characterizing many of the changes in protein composition, when coupled with specific histological-type staining reactions. The results obtained here by using extracts from cells at different stages of growth and sporulation are consistent with observations from other laboratories where extensively purified and highly characterized enzymes have been studied. In several instances, the histochemical reactions can be associated with a specific enzymatic function and appear to indicate the presence of multiple molecular forms. In other instances, the data cannot be evaluated in terms of known enzyme function because the specificity of the histochemical analysis is not certain. However, the assays described permit monitoring of electrophoretic changes at the level of individual proteins within sporulating cultures. The results suggest that B. subtilis may contain two "hexokinase-like" enzymes which cease to function before sporulation is initiated. Aldolase and alanine dehydrogenase are detectable as single bands of enzyme activity during vegetative growth but as multiple molecular forms once sporulation has been initiated. Reduced nicotinamide adenine dinucleotide dehydrogenase activity is represented by an entire family of reactive species in these crude extracts, which undergo multiple changes during the early stages of sporulation. Tricarboxylic acid cycle dehydrogenase enzymes and those bands having esterase activity on alpha-naphthyl acetate show detectable changes in specific activity after cessation of exponential growth. Glucose dehydrogenase is not detectable until the sequence of changes leading to spore formation has progressed for 4 or 5 hr.

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Spectinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc 1-11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1-11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.

Mutation affecting expression of spectinomycin resistance in Bacillus subtilis.

A mutation that affects the expression of spectinomycin resistance in a spectinomycin-resistant (spcA), conditionally asporogenic strain of Bacillus subtilis has been designated srm (spectinomycin resistance modifier). This mutation resulted in altered colony morphology and increased growth rate and sporulation efficiency in the presence of spectinomycin.

Bacillus subtilis deoxyribonucleic acid gyrase.

Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics. The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B. subtilis and was functionally characterized in vitro. The genetic loci nalA and novA but not novB were shown to code for portions of the functional gyrase. Enzyme from the antibiotic-resistant mutants was resistant to the drug in vitro. The most striking observation was the remarkable similarity between the B. subtilis enzyme and other DNA gyrases, especially with respect to the oxolinic acid-induced DNA cleavage in the presence of sodium dodecyl sulfate. All of the enzymes appeared to possess the same specificity of cutting sites regardless of the source or type of DNA used. This result implies that gyrase binding to DNA is highly specific.

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.

Development of competence in the Bacillus subtilis transformation system.

Competence in Bacillus subtilis, assayed by the ability of cells to be transformed with bacterial deoxyribonucleic acid (DNA) or transfected by phage DNA, has been shown to occur in a single semisynthetic medium with peak activity occurring 3 hr after the cessation of logarithmic growth. No step-down conditions or culture manipulations were necessary for routine transfection of 1% of the population. The results demonstrate that bacteriophage DNA is a valid assay for studying the development of competence in B. subtilis. Predictions of workers using transforming bacterial DNA, who have suggested that competence in B. subtilis is associated with a specific phase of growth, are substantiated. The peak of competence is not affected by marked differences in the rate of growth during the logarithmic phase. The effect on development of competence by this procedure of some components (including casein hydrolysate, tryptophan, and histidine) which were routinely included in the transformation medium by other investigators has been determined by use of infectious phage DNA as an assay. We have demonstrated that tryptophan, as well as histidine, increases the transformation frequency-even in strains which do not have auxotrophic demands for these components. Glutamic acid and alanine depress optimal levels of transfection.