Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Genome sequences of Mycoplasma alligatoris A21JP2T and Mycoplasma crocodyli MP145T.

Mycoplasma alligatoris and Mycoplasma crocodyli are closely related siblings, one being highly virulent and the other relatively attenuated. We compared their genomes to better understand the mechanisms and origins of M. alligatoris' remarkable virulence amid a clade of harmless or much less virulent species. Although its chromosome was refractory to closure, M. alligatoris differed most notably by its complement of sialidases and other genes of the N-acetylneuraminate scavenging and catabolism pathway.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

The complete sequence of the mucosal pathogen Ureaplasma urealyticum.

The comparison of the genomes of two very closely related human mucosal pathogens, Mycoplasma genitalium and Mycoplasma pneumoniae, has helped define the essential functions of a self-replicating minimal cell, as well as what constitutes a mycoplasma. Here we report the complete sequence of a more distant phylogenetic relative of those bacteria, Ureaplasma urealyticum (parvum biovar), which is also a mucosal pathogen of humans. It is the third mycoplasma to be sequenced, and has the smallest sequenced prokaryotic genome except for M. genitalium. Although the U. urealyticum genome is similar to the two sequenced mycoplasma genomes, features make this organism unique among mycoplasmas and all bacteria. Almost all ATP synthesis is the result of urea hydrolysis, which generates an energy-producing electrochemical gradient. Some highly conserved eubacterial enzymes appear not to be encoded by U. urealyticum, including the cell-division protein FtsZ, chaperonins GroES and GroEL, and ribonucleoside-diphosphate reductase. U. urealyticum has six closely related iron transporters, which apparently arose through gene duplication, suggesting that it has a kind of respiration system not present in other small genome bacteria The genome is only 25.5% G+C in nucleotide content, and the G+C content of individual genes may predict how essential those genes are to ureaplasma survival.

Sequencing multimegabase-template DNA with BigDye terminator chemistry.

Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of >700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial genome sequencing projects without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.

Phylogenetic analysis of the 16S-23S rRNA intergenic spacer regions of the genus Ureaplasma.

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of six Ureaplasma species were determined following amplification by polymerase chain reaction. The hypothetical secondary structure of the spacer regions was divided into four domains. Domains I and III were highly conserved among different Ureaplasma species but domains II and IV were variable. The intergenic spacer regions of ureaplasmas lacked spacer tRNA genes. Phylogenetic relationships among the Ureaplasma species indicated that the evolutionary history of the Ureaplasma species follows that of the host animal species, suggesting coevolution between ureaplasmas and animals.

Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma pulmonis: evidence for DNA inversion as a mechanism for V-1 variation.

Although the variation of V-1 antigens of Mycoplasma pulmonis has been correlated with variable expression of the cytadherence properties of this organism and has been implicated as a virulence determining factor in M. pulmonis-induced murine respiratory disease, the precise function of these antigens remains unknown. We have cloned and characterized genes encoding V-1 from two M. pulmonis UAB CT V-1 variants that differ in hemadsorption properties. A comparison of the nucleotide sequences revealed that these two variant genes were identical in the 5'-most 724 nucleotides. Regions of extensive divergence that contained repeated sequences were found 3' to this conserved region. On the basis of their deduced amino acid sequences, one variant expressed a V-1 protein of 94.2 kDa presumptively containing 40 repeats of 17 amino acids and the other expressed a protein of 27.4 kDa consisting 2 direct, noncontiguous 9-amino-acid repeats. These general properties, as well as the presence of a prokaryotic lipoprotein acylation sequence (L-X-Y-C), indicated that the genes encoding V-1 were similar in structure to genes encoding other mycoplasma surface lipoproteins. Further analysis of sequences flanking these genes revealed that these variants arose via an inversion event which provided an interchange of the two variable regions as well as for the conserved region of these genes and immunoblot analyses using rabbit polyclonal antibodies specific for synthetic peptides derived from the sequences of the different variable regions indicated that DNA inversion acted as a switch which allowed only one of the two different genes to be expressed at any given time. This inversion model clearly provides a mechanism by which M. pulmonis can alter its surface architecture and also strongly suggests that the as-yet-undefined function of V-1 residues in the variable carboxy region of these proteins.

Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigen size variation.

Ureaplasma urealyticum is a common commensal of the female lower urogenital tract, yet it has been shown to be an important cause of chorioamnion infection, respiratory and central nervous system disease, and death in premature infants. It has been suggested that only certain serovars are capable of producing invasive disease. However, we previously showed that many serotypes are invasive and that perhaps antigen variability and host factors are more important determinants of ureaplasma infections than are different serotypes per se. The molecular characterization in this report describes a mechanism available to ureaplasmas for producing antigen variation. That antigen, designated MB and previously identified on U. urealyticum, contains serovar-specific and cross-reactive epitopes, is produced both in vitro and in vivo, is a predominant antigen recognized during ureaplasma infections of humans, undergoes a high rate of size variation in vitro, and is size variable on invasive ureaplasma isolates. In the present study, we cloned and sequenced the gene of the MB antigen from serovar 3, the serovar most commonly isolated from humans. The 3' two-thirds of the gene was shown to contain identical 18-nucleotide tandem repeats. PCR analysis and direct sequencing of two variants indicated that alterations within this repeat region are responsible for the size variation of the MB antigen. Intact recombinant serovar 3 MB antigen and truncated products, expressed by coupled in vitro transcription and translation of the cloned gene, were immunoprecipitated by both a serovar-specific monoclonal antibody and the serum of a U. urealyticum-infected patient, and these results identified the repeat region of the MB antigen as serovar defining. Resolution of the precise amino acids responsible for specific epitopes and characterization of similar genes in the other serovars should yield reagents useful in elucidating the role of antigen size variants in disease production and the role of specific antibody in protection from ureaplasma disease.

Ureaplasma urealyticum biovar specificity and diversity are encoded in multiple-banded antigen gene.

Ureaplasma urealyticum is a commensal organism of the lower genital tract of females, but in a subpopulation of individuals, it can invade the upper genital tract. It is a significant cause of chorioamnionitis and neonatal morbidity and mortality. There are 14 recognized serovars of U. urealyticum; these can be divided into two distinct clusters or biovars. Biovar 1 is composed of serovars 1, 3, 6, and 14, Biovar 2 is composed of serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13. We previously identified a surface antigen, the multiple-banded (MB) antigen, which contains both serovar-specific and cross-reactive epitopes. Genotypic characterization of the C-terminal region of the MB antigen of serovar 3 indicates that serovar specificity and MB antigen size variation reside in that domain. In the present study, we used PCR analysis with primers derived from the serovar 3 MB antigen gene DNA sequence to determine if the MB antigen gene was present in the remaining 13 reference serovars as well as in invasive clinical isolates. The results indicated that not only was the MB antigen gene present in all serovars but that the genes' 5' regions were markers of biovar specificity and diversity. Further analysis of this region should reveal the phylogenetic relationship among serovars of U. urealyticum and, possibly, their invasive potential.