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.

Contribution of serum inflammatory markers to changes in bone mineral content and density in postmenopausal women: a 1-year investigation.

Bone formation and resorption are influenced by inflammatory processes. We examined the relationships among inflammatory markers and bone mineral content (BMC) and density (BMD) and determined the contribution of inflammatory markers to 1-yr changes in BMC and BMD in healthy postmenopausal women. This analysis included 242 women at baseline from our parent Soy Isoflavones for Reducing Bone Loss project who were randomly assigned to 1 of 3 treatment groups: placebo, 80 mg/d soy isoflavones, or 120 mg/d soy isoflavones. BMD and BMC from the lumbar spine (LS), total proximal femur (hip), and whole body were measured by dual energy X-ray absorptiometry and the 4% distal tibia by peripheral quantitative computed tomography. Serum inflammatory markers (C-reactive protein, interleukin [IL]-1 beta, IL-6, tumor necrosis factor-alpha [TNF-alpha], and white blood cell count [WBC]) were measured at baseline, 6, and 12 mo. Because of attrition or missing values, data analysis at 12 mo includes only 235 women. Significant associations among IL-6, TNF-alpha, and WBC were observed with percent change in LS, hip, and whole body BMC and BMD. Multiple regression analysis indicated that in combination inflammatory markers accounted for 1.1-6.1% of the variance to the observed 12-mo changes in BMC and BMD. Our results suggest that modifying inflammatory markers, even in healthy postmenopausal women, may possibly reduce bone loss.

Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins.

The ability of some microorganisms to rapidly alter the expression and structure of surface components reflects an important strategy for adaptation to changing environments, including those encountered by infectious agents within respective host organisms. Mycoplasma hyorhinis, a wall-less prokaryotic pathogen of the class Mollicutes, is shown to undergo high-frequency phase transitions in colony morphology and opacity and in the expression of diverse lipid-modified, cell-surface protein antigens. These proteins spontaneously vary in size, contain highly repetitive structures, and are oriented with their carboxyl-terminal region external to the membrane. Thus, mycoplasma membrane lipoproteins generate microbial surface diversity and may be part of a complex system that controls interactions of these organisms with their hosts.

Vesicular stomatitis virus-infected L1210 murine leukemia cells: increased immunogenicity and altered surface antigens.

Homogenates of L1210 cells infected in vitro with vesicular stomatitis virus (VSV) were immunogenic agains a tumor graft of 100 times the LD50 dose of L1210 cells" whereas those of uninfected cells were not. The immunogenicity of intact X-irradiated L1210 cells was distinguishable from that of VSV-infected cell homogenates on the basis of the susceptibility of immunogenicity to experimental procedures used in preparation of the immunogenic homogenates: Homogenization of intact X-irradiated cells or their infection with VSV prior to irradiation led to loss of immunogenicity. In addition, uninfected cell homogenates were not made immunogenic nor was the immunogenicity of VSV-infected cell homogenates eliminated by X-irradiation. At the time of tumor challenge, sera from mice that were effectively immunized with VSV-infected cell homogenate showed a high VSV-neutralizing titer but no complement-dependent cytotoxicity for L1210 cells. Quantitative absorption studies demonstrated that VSV infection led to a marked reduction in L1210 surface antigens recognized by cytotoxic alloantibody; spatial association between these antigens and VSV antigens was not demonstrable on VSV-infected cells. Antigens recognized by heterologous antiserum to L1210 cells were also reduced following VSV infection.

Adaptive surface variation in mycoplasmas.

Mycoplasmas excel as infectious agents, despite their very small genomes. In one mycoplasma species, adaptive flexibility is enhanced by an elegant genetic system that diversifies the membrane surface through a set of variable lipoproteins (Vlps). A family of vlp genes supplies divergent coding sequences and undergoes high-frequency mutations, thus creating large repertoires of surface mosaics and structural variants.

A Mycoplasma hyorhinis protein with sequence similarities to nucleotide-binding enzymes.

We have determined the nucleotide (nt) and deduced amino acid (aa) sequence of a unique 115-kDa Mycoplasma hyorhinis protein (P115) with an N-terminal region containing a highly conserved consensus sequence characteristics of nt-binding domains of several ATPase and GTPase enzymes. However, P115 lacked additional conserved features characteristic of some classes of nt-binding proteins. Based on the hydropathy profile of the deduced aa sequence, the absence of a leader peptide, its exclusive partitioning into the hydrophilic phase during Triton X-114 phase fractionation of M. hyorhinis, and immunofluorescence analysis indicating no surface-exposed domains, it was concluded that P115 is a cytoplasmic protein lacking intrinsic membrane interaction. M. hyorhinis P115 appears to be a species-specific protein, since it was not detected in any other mycoplasmal or bacterial species examined with specific antibody or genomic probes. Since genetic systems for direct mutational analysis are currently unavailable in this organism, sequence analysis provides critical information in establishing the possible function of this protein. Moreover, the nt sequence encoding P115 reported here supports a previously proposed model, based on synthesis of P115-related proteins in Escherichia coli, suggesting that multiple polypeptide products can be generated from mycoplasma genes by promiscuous translation initiation in this heterologous expression system.

Immunologic cross-reaction between HIV type 1 p17 and Mycoplasma hyorhinis variable lipoprotein.

Monoclonal antibodies directed against the HIV-1 matrix protein p17 that react with a component present on the surface of HIV-1-infected cells have previously been described. In this study we show that one of these monoclonal antibodies binds to persistently HIV-1-infected cell lines that are coinfected with Mycoplasma hyorhinis, but not to cell lines that are uninfected with mycoplasma. Mycoplasma-infected cells secrete HIV-1 at a higher rate, have a slight increase in cell surface expression of gp120 and gp41, and are less sensitive to immunotoxins than uninfected cells. The anti-p17 antibody binds to a protein of M. hyorhinis grown in cell-free culture. The variable expression and size of the protein among strains is typical of the variable lipoprotein (Vlp) system of M. hyorhinis. Confirmation of the reactivity of the antibody with a Vlp was provided by demonstrating its specific binding to recombinant VlpF expressed in E. coli, and to a synthetic peptide representing the carboxy-terminal region of VlpF, but not to other recombinant Vlp products or peptides. This is a true cross-reaction because the antibody also binds to recombinant p17 expressed in E. coli and the binding is inhibited by the VlpF peptide. These analyses highlight the potential of mycoplasma contamination of tissue culture cell lines to cause anomalous results. With regard to HIV-1, mycoplasma infection of cells results in increased rates of virus secretion, and introduces a potential confounding immunologic cross-reaction as well. The existence of a cell surface form of p17 is unlikely.

Isolation of Wilms' tumor antigens by chelation.

A procedure for ethylenediaminetetraacetate extraction of minced Wilm's tumor was assessed as a method for isolating Wilm's tumor antigens. An antigen was detected by immunodiffusion using an adsorbed antiserum to this extract. This antigen was also found in ethylenediaminetetraacetate extracts of in vitro cultures of nephroblastoma cells.

The anatomy of the metacarpo-phalangeal joints, with observations of the aetiology of ulnar drift.

One hundred normal fingers were dissected and arthrographs obtained by injection of chromopaque-gelatin mixture, allowing comparison between the radiographic and macroscopic configuration of the synovial capsule. Synovial recesses protruding from each side of every metacarpo-phalangeal joint were found in relation to the collateral ligaments and corresponding exactly with the site of radiological erosions. A group of bursae lying on the superficial aspect of collateral ligaments were also demonstrated. A rudimentary intra-articular meniscus was found. The results of examination of the insertions of the interossei showed differences from traditional descriptions. The cause of rheumatoid deformity was suggested to be the rheumatoid process arising in the lateral recesses and lateral bursae, weakening the collateral ligaments, which give way in the directions of the deforming forces. These are derived from the long flexor tendons, which were shown to exert an ulnar and volar strain on the metacarpo-phalangeal joint of every finger during grip.

Cine radiography in cervical spondylosis as a means of determining the level for anterior fusion.

A series of 75 patients who had undergone anterior cervical fusion between 1965 and 1977 were reviewed. The patients were divided into two groups: those in Group A had had the level of fusion indicated by cine radiography, whereas in Group B the level had been determined by plain radiographs and clinical symptoms and signs. Results showed that cine radiography was the more accurate diagnostic technique. Accurate diagnosis of the level to be fused, the careful clinical selection of patients and sound bony union were found to be vital to the success of anterior cervical fusion. The incidence of pseudarthrosis was significant in single-level fusions and was even greater in double-level fusions and in patients with a history of trauma, especially whiplash injuries. It was rare to develop recurrence of symptoms in adjacent levels after fusion of a level localised by cine radiography.

Productive murine leukemia virus (MuLV) infection of EL4 T-lymphoblastoid cells: selective elevation of H-2 surface expression and possible association of Thy-1 antigen with viruses.

This study evaluated differences in the expression of three surface antigens (H-2Kb, H-2Db, and Thy 1.2) of two EL4 T-lymphoblastoid cell lines. The two cell lines, EL4 (G-) which is not virus infected and the MuLV-producing subline EL4G +, had the same cytographic size distribution and a doubling time of twenty-four hours. Reflective of the morphological similarities, the expression of the T-cell specific alloantigen Thy 1.2 for the two lines did not differ. In contrast to the Thy 1.2 expression, antibody-detectable H-2Kb and H-2Db activity was much greater on the MuLV-producing EL4G + subline as indicated by fewer cells needed to remove 50% of the cytotoxic activity of the antibody ( AD50 ). Comparison of the EL4(G-) AD50 / EL4G +AD50 ratio indicated an increase in H-2b antigen expression on EL4G + of 15.6 fold for H-2Kb and 7.81 fold for H-2Db. To investigate the possibility of a selective association between MuLV and EL4G + H-2 antigens, the specific activity for H-2Kb, H-2Db and Thy 1.2 of EL4G + membrane and MuLV preparations obtained from EL4G + cultures was compared. The relative proportion of (Thy 1: H-2Kb: H-2Db) in membrane preparation was (1.14:2.45:1.95) compared to the proportion (0.31:3.15:0.95) in virus preparations. Although the amount of Thy-1 activity was markedly elevated in the virus preparation, H-2b antigenic activities were not significantly different in the two preparations. These data are consistent with an active association of lymphoid surface glycoproteins and MuLV, and suggest an association between productive MuLV infection and an increase in the expression of H-2 antigens in the EL4G + cell cultures.

Antigen expression from cloned genes of Mycoplasma hyorhinis: an approach to mycoplasma genomic analysis.

Cloned DNA segments of the Mycoplasma hyorhinis genome have been identified by specific immunological detection of gene products expressed in Escherichia coli. Individual, distinct segments collectively representing greater than 10% of the genome each synthesize multiple proteins, some of which are immunogenic. Molecular genetic and immunologic tools have identified mycoplasma genomic fragments useful in 1) generating and analyzing specific, isolated mycoplasma gene products, 2) studying structure and expression of specific mycoplasma genes, and 3) generating specific gene probes. Immunological detection provides the potential for preselection of specific genes of interest.

The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation.

Isogenic populations of Mycoplasma hyorhinis undergo in vitro high-frequency phase variation in the expression of surface lipoproteins; these products also vary markedly in size through changes in periodic protein structure (R. Rosengarten and K.S. Wise, Science 247:315-318, 1990). In this report, we rigorously define three distinct translation products comprising the Vlp (variable lipoprotein) system of M. hyorhinis SK76 and establish parameters of Vlp structural diversity and expression that distinguish the Vlp system from previously described examples of antigenic variation. VlpA, VlpB, and VlpC are prominent amphiphilic membrane lipoproteins characterized by detergent-phase fractionation and metabolic labeling with [35S]cysteine and [3H]palmitate. VlpA is distinguished from VlpB and VlpC by its selective labeling with [35S]methionine; VlpB and VlpC are distinguished by specific epitopes defined by surface-binding monoclonal antibodies (MAbs); a third MAb defines a surface epitope shared by VlpB and VlpC (but absent from VlpA). Each Vlp displays 12 to 30 spontaneous size variant forms comprising a periodic ladder that could also be generated by partial trypsin digestion of individual Vlp size variants. Different periodic intervals within VlpB and VlpC further distinguish these two products structurally. Mycoplasma colony opacity correlates inversely with Vlp size. Each Vlp undergoes independent, oscillating high-frequency phase variation in isogenic populations and can be expressed individually or concomitantly with other Vlps in a noncoordinate manner. All seven possible combinations of these three products were observed; however, no variants were found that lacked a Vlp. High-frequency size variation of each Vlp superimposed on combinatorial diversity in Vlp expression yields greater than 10(4) possible structurally distinct Vlp mosaics, of which 104 were documented along with 24 of 42 possible transitions among the seven Vlp combinations. In addition to these features, VlpA, VlpB, and VlpC were specifically recognized by serum antibodies from swine with experimental M. hyorhinis SK76-induced arthritis, indicating expression and immunogenicity of Vlps in the natural host. The structure and variation of Vlps and their known involvement in MAb-mediated modulation of mycoplasma-infected host cell properties and mycoplasma killing are discussed in relation to the surface architecture and adaptive potential of the wall-less mycoplasmas.

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.

Identification of intrinsic and extrinsic membrane proteins bearing surface epitopes of Mycoplasma hyopneumoniae.

A library of monoclonal antibodies (McAbs) raised against Mycoplasma hyopneumoniae was screened to select McAbs that bound both to surface epitopes on intact organisms and to corresponding proteins identified by immunoblot analysis. Four proteins (indicated by MW in kilodaltons) were established as surface antigens: p65, p50, p44 and p41. Triton X-114 detergent phase fractionation of whole organisms clearly distinguished p65, p50 and p44 as hydrophobic integral membrane proteins, whereas p41 was identified as a hydrophilic surface protein apparently extrinsic to the membrane. This technique also provided a rapid and efficient method for isolating the relatively small number of hydrophobic proteins associated with M. hyopneumoniae. Preliminary evidence suggests that p65, p50 and p44 are linked covalently to lipids, which may be important in dictating their interaction with the membrane. Immunoblot analysis of antibodies from swine immunized with M. hyopneumoniae also suggests that p65 and p44 may be "immunodominant" antigens in this host.