The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.

A physical map of the chromosome of Lactococcus lactis subsp. lactis DLl1 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one-and twodimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DLll chromosome. By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed-and fixed-field digestion patterns, six putative rRNA operons were identified in L. lactis subsp. lactis DLll and placed on the map of the chromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced.The gram-positive bacteria Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris (formerly Streptococcus lactis and S. cremoris, respectively) are used extensively as starter organisms for the manufacture of cheese. Interest in the molecular biology of these organisms is heightened by their industrial importance and because they and their metabolic products are generally regarded as safe for human consumption.Analysis of the L. lactis genome has been restricted to measurements of its base composition (36 to 38% G+C [16]) and the use of renaturation kinetics to determine its size (2.75 to 3.1 megabase pairs [Mbp] [13]). Preliminary results from pulsed-field gel electrophoresis (PFGE) indicate somewhat smaller genome sizes of 2.0 to 2.7 Mbp for a number of strains of L. lactis (20,33).Most strains of L. lactis contain four or more different plasmids. The properties of a number of these plasmids have been reported, and a number of plasmid-borne genetic determinants have been characterized at the molecular level (4,18,21,29). In contrast, there are only a few reports of the cloning of chromosomal DNA (26, 35) and a genetic map of the lactococcal chromosome has not been developed. Because of the paucity of chromosomal markers and the unavailability of suitable techniques for transferring chromosomal genes within the lactococci, PFGE offers the most promise for analyzing and mapping the lactococcal chromosome.

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of 14 C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides . Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5′-monophosphate to adenosine 5′-monophosphate via the intermediate inosine 5′-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.The use of pulsed-field gel electrophoresis for the separation of large restriction fragments has made possible the construction of physical maps for several procaryotic genomes, including two large colony (LC) strains of Mycoplasma mycoides subsp. mycoides, the Y goat strain, and GC1176-2 (10, 19a, 20). These show congruence in the gene loci and many of the mapped restriction sites when three divergences totalling 180 kbp are allowed for in the larger genome of GC1176-2. M. mycoides subsp. mycoides also includes small colony (SC) strains, and the subspecies is part of a larger grouping of ruminant pathogens called the M. mycoides cluster (4). Here, we compare the map for M. mycoides subsp. mycoides Y goat with the genomic restriction maps and placement of various gene loci for four SC strains of M. mycoides subsp. mycoides and one strain, PG50, of bovine serogroup 7, another member of the cluster.M. mycoides subsp. mycoides SC strains PG1, KH3J, Gladysdale, and V5 were from the culture collection of G. Cottew, whereas PG50 was obtained from C. Christiansen. Preparation and analysis of DNA in agarose to construct restriction maps were as previously described (3,(8)(9)(10). Gene loci were detected by probing Southern blots with cloned DNAs (Table 1). Sizes of fragments up to 500 kbp were measured against bacteriophage A DNA multimers as markers, whereas for larger fragments the markers were yeast chromosomal DNA molecules (Bio-Rad Laboratories) and BssHII and ApaI digests of DNA from M. mycoides subsp. mycoides Y goat (10) and GC1176-2 (19a). The sizes of the single BssHII fragments, representing the complete genomes, from the DNA of the SC strains were in reasonable agreement with the sums of the sizes of the fragments in the other digests. From these values, the sizes of the genomes fell in a range of about 1,230 to 1,280 kbp compared with values of 1,200 and 1,380 kbp for map sizes of LC M. mycoides Y goat and GC1176-2, respectively. The size of the PG50 genome was not measured as a single fragment, but the sums of the fragment sizes for each of the digests were in reasonable agreement with the rounded average value of 1,040 kbp shown in Fig.