Deoxyribonucleic Acid Homology and Relative Genome Size in Mycoplasma

The deoxyribonucleic acid homologies of Mycoplasma laidlawii type A and type B, M. pulmonis (#47 and #63), and M. hominis were determined by membrane methodology. The homology data revealed a difference in genome size between M. laidlawii type A and type B. This difference also held with stringent conditions of annealing (high temperature). Little or negligible homology was shown to exist between the M. laidlawii strains type A and type B and M. pulmonis strains 47 and 63 and M. hominis. M. hominis showed less than 10% homology to the M. pulmonis and M. laidlawii strains. Neither of the M. laidlawii strains showed more than 2% annealing to the M. pulmonis strains. Reaction rate studies are suggested as a means of demonstrating the phylogenetic relationship between the Mycoplasma and other microorganisms.     

Binding of lectins to membranes of mycoplasmas from aging cultures

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.     

Differences in accumulation of radiolabeled amino acids and polyamines by Mycoplasma and Acholeplasma species

Four species in the order Mycoplasmatales, Mycoplasma capricolum, Mycoplasma hominis, Mycoplasma arginini, and Acholeplasma laidlawii, were compared for their ability to accumulate radiolabeled amino acids and polyamines. The use of a novel high-molecular-weight (HMW) medium, from which molecules of less than 12,000 molecular weight had been removed by extensive dialysis, allowed us to discern significant differences among the species in their relative accumulations of [3H]methionine and [3H]leucine and of [3H]spermidine and [3H]putrescine. Accumulation of radiolabeled amino acids in control low-molecular-weight (LMW) medium was small (0.2 to 2% of the label), and the species did not differ in their proportional accumulations of methionine and leucine. Accumulation of methionine was significantly enhanced (5- to 12-fold) in all species in HMW medium. In contrast, leucine accumulation was enhanced sevenfold for A. laidlawii but only twofold for M. hominis and M. capricolum in HMW medium. The nonglycolytic species, M. hominis and M. arginini, accumulated radiolabeled putrescine and spermidine in both media, whereas the glycolytic species, M. capricolum and A. laidlawii, accumulated only radiolabeled spermidine. The ability to accumulate putrescine appeared to be a differential characteristic for nonfermentative, arginine-utilizing mycoplasmas. HMW medium was much more effective than LMW medium for use in radiolabeling M. capricolum proteins with [35S]methionine.     

Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol

Smith, Paul F. (University of South Dakota, Vermillion), and Carl V. Henrikson. Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol. J. Bacteriol. 91:1854-1858. 1966.-Compounds which inhibit enzymatic reactions in the biosynthetic pathway to carotenoids inhibited growth of a sterol-nonrequiring species, Mycoplasma laidlawii, strain B, and M. hominis, strain 07. Since M. hominis lacks the enzymes for polyterpene biosynthesis, the inhibitory compounds must act also at other sites. Most inhibitors exerted a lytic effect at bactericidal levels. The inhibition of M. laidlawii is reversed by exogenous cholesterol. M. laidlawii exhibited a greatly increased content of cholesterol and a greatly decreased content of carotenoids when grown in the presence of phenethylbiguanide and cholesterol. These results are considered as further evidence for a common function for sterols and carotenols in Mycoplasma.     

Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure.     

Cloning and expression of the gene for the mycoplasma key division protein FtsZ in Escherichia coli]

Gene ftsZ responsible for division of bacterial cells was revealed in most prokaryote groups. A 520-bp fragment of the ftsZ gene was amplified on the template of A. laidlawii DNA using degenerate primers. This fragment was sequenced and served as a hybridization probe for cloning of the full-sized copy of the A. laidlawii ftsZ gene. The amplified fragment was cloned in a pGEX3X vector and expressed in E. coli cells. Polyclonal antibodies derived from the chimeric polypeptide containing a fragment of A. laidlawii FtsZ protein interacted only with the A. laidlawii protein with molecular mass of 40 kDa. Comparison of nucleotide sequences of the ftsZ-gene region of A. laidlawii and other bacterial species showed that they were highly homologous in A. laidlawii, E. coli, and Bac. subtilis, while low homology was revealed between the A. laidlawii sequence and those of the members of the genus Mycoplasma. Analysis of the ftsZ-gene nucleotide sequences is suggested as a means to study the evolutionary relatedness of prokaryotes.     

Cultivation of Mycoplasmas on Cellulose Ester Substrates

The ability of mycoplasmas to grow on cellulose ester substrates was evaluated. Mycoplasma pneumoniae, M. hominis, M. arthritidis, M. gallisepticum, and Acholeplasma laidlawii grew on Millipore (mixed cellulose ester) filters and Sepraphore III (cellulose polyacetate) membranes.     

Inactivation of the vascular permeability-increasing activity of bradykinin by mycoplasmas

Mycoplasma pneumoniae, M. genitalium, M. fermentans, M. hominis, M. salivarium, M. orale, Ureaplasma urealyticum and Acholeplasma laidlawii inactivated the vascular permeability-increasing activity of bradykinin when the mixture of bradykinin and mycoplasma cells was injected after incubation at 37 degrees C for 1 h. Cell components responsible for inactivation of the activity of bradykinin were found to be arginine-specific aminopeptidase and carboxypeptidase.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment.

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The phenotype of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Cloning and DNA sequence of a mycoplasmal recA gene.

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.     

The photosensitizing activity of haematoporphyrin on mollicutes

The photosensitizing activity of haematoporphyrin (HP) on Mycoplasma hominis and Acholeplasma laidlawii was studied as a function of the phase of growth and the amount of sterols in the cell membrane. Less HP was bound to cells when the membrane had a high sterol content. Both strains in the exponential but not in the stationary phase of growth were sensitive to HP treatment (above 1 microgram ml-1) in the dark. Visible light irradiation of HP-loaded cells caused in all cases a decrease of cell survival, with concomitant changes in the pattern of membrane proteins that suggested protein-protein cross-linking, and the appearance of ultrastructural alterations (rounded and lysed cells); the photosensitivity was indirectly related to the sterol content of the cell membrane. On the whole, our findings suggest that the cell membrane is a major target for HP photosensitization of mycoplasma cells.     

Protease domain of human ADAM33 produced by Drosophila S2 cells

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].     

Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.     

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.     

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.