Nutritional requirements of two flower spiroplasmas and honeybee spiroplasma.

A chemically defined medium (CC-494) was used to study the nutritional requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, the honeybee spiroplasma could ferment trehalose, FS (SR-3) could ferment sucrose, and S. floricola could ferment trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in their requirements of amino acids for growth. S. floricola was the only strain that utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base (either free base or ribonucleoside) for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and bovine serum albumin were essential to all three spiroplasmas. Palmitic acid, which was nonessential, promoted growth significantly.     

Vitamin requirements of three spiroplasmas.

A chemically defined medium (CC-494M) was used to study the vitamin requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Nicotinic acid and riboflavin were essential to spiroplasma growth. Nicotinamide could substitute for nicotinic acid. Populations of S. floricola, FS (SR-3), and HBS (AS-576) reached 3.2 X 10(9), 1.96 X 10(10), and 6.1 X 10(9) CFU/ml, respectively, when nicotinic acid (0.036 mg/liter) and riboflavin (0.014 mg/liter) were supplied.     

Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.     

Procaryotic and eucaryotic traits of DNA methylation in spiroplasmas (mycoplasmas).

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Simplified media for spiroplasmas associated with tabanid flies

Traditionally, isolation, maintenance, and testing of Spiroplasma species (Mollicutes: Entomoplasmatales) from horse flies (Tabanus spp.) and deer flies (Chrysops spp.) (Diptera: Tabanidae) have been accomplished in the complex M1D medium. A relatively inexpensive, simplified medium for tabanid spiroplasmas could expedite procedures that require large quantities of growth medium. Nine strains of spiroplasmas, eight from tabanids and one from mosquitoes, were cultured in three simplified broth media, R2, R8-1, and C-3G, and in M1D. There was no significant difference in the rate of spiroplasma growth in M1D and the three simplified media. R2 medium supported the growth of tabanid spiroplasmas more consistently and with better morphology through 10 subcultures than did the other simplified media. Primary isolations were made in R2 medium from tabanids collected (i) in Georgia, U.S.A., with 10 isolations from 10 flies and (ii) in coastal Costa Rica, with isolation rates of 70% (28/40) and 73% (27/37), respectively, for R2 and M1D. Of the seven group VIII field isolates from Costa Rica, four were capable of sustained growth in R2, and three were triply cloned in this simplified medium. These results suggest that the simplified medium R2 is suitable for many procedures with tabanid spiroplasmas.     

Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture.

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.     

Neokomagataea gen. nov., with descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the α-Proteobacteria

Isolates AH11(T) and AH13(T) were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11(T) were 95.7-92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11(T) was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11(T) and AH13(T) and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C(18:1)ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C(18:1)2OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11(T) (=BCC 25710(T)=NBRC 106555(T)), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13(T) (=BCC 25711(T)=NBRC 106556(T)), which has 51.2 mol % G+C.     

The utilization of sugars and starch as carbon sources by sugarcane cell suspension cultures

Best growth of sugarcane cell suspensions on sugar substrateswas obtained with raffinose, followed closely by sucrose. Growthon glucose, fructose, cellobiose, melibiose and trehalose wasslightly below that on sucrose. Pentoses have been reportedpreviously not to be utilized by plant tissues in culture-ribosesupported growth of the sugarcane cells. Starch supported growthas well as did sucrose. This support is due to the secretionof an -amylase by the sugarcane cells in culture. ¹Published with the approval of the Director as Paper No. 239in the Journal Series of the Experiment Station, Hawaiian SugarPlanters' Association, Honolulu, Hawaii, U.S.A. (Received October 3, 1969; )     

Redundancy in periplasmic binding protein-dependent transport systems for trehalose,sucrose, and maltose in Sinorhizobium meliloti

We have identified a cluster of six genes involved in trehalose transport and utilization (thu) in Sinorhizobium meliloti. Four of these genes, thuE, -F, -G, and -K, were found to encode components of a binding protein-dependent trehalose/maltose/sucrose ABC transporter. Their deduced gene products comprise a trehalose/maltose-binding protein (ThuE), two integral membrane proteins (ThuF and ThuG), and an ATP-binding protein (ThuK). In addition, a putative regulatory protein (ThuR) was found divergently transcribed from the thuEFGK operon. When the thuE locus was inactivated by gene replacement, the resulting S. meliloti strain was impaired in its ability to grow on trehalose, and a significant retardation in growth was seen on maltose as well. The wild type and the thuE mutant were indistinguishable for growth on glucose and sucrose. This suggested a possible overlap in function of the thuEFGK operon with the aglEFGAK operon, which was identified as a binding protein-dependent ATP-binding transport system for sucrose, maltose, and trehalose. The K(m)s for trehalose transport were 8 +/- 1 nM and 55 +/- 5 nM in the uninduced and induced cultures, respectively. Transport and growth experiments using mutants impaired in either or both of these transport systems show that these systems form the major transport systems for trehalose, maltose, and sucrose. By using a thuE'-lacZ fusion, we show that thuE is induced only by trehalose and not by cellobiose, glucose, maltopentaose, maltose, mannitol, or sucrose, suggesting that the thuEFGK system is primarily targeted toward trehalose. The aglEFGAK operon, on the other hand, is induced primarily by sucrose and to a lesser extent by trehalose. Tests for root colonization, nodulation, and nitrogen fixation suggest that uptake of disaccharides can be critical for colonization of alfalfa roots but is not important for nodulation and nitrogen fixation per se.     

Serological analysis of a new group of spiroplasmas

Seven spiroplasmas recovered from insects and flower surfaces in widely separated geographical areas were analyzed serologically by metabolism inhibition, enzyme-linked immunosorbent, and growth inhibition tests. Although the seven organisms were serologically interrelated, they were not identical. The seven spiroplasmas in this cluster did not show serological cross-reactions to other established serogroups of spiroplasmas, including theSpiroplasma citri complex. Results of the serological analyses correlate well with genome size differences recently documented for this group of spiroplasmas and support a recently proposed classificatin scheme.     

Dietary nutrient levels regulate protein and carbohydrate intake,gluconeogenic/glycolytic flux and blood trehalose level in the insect<Emphasis Type="Italic"> Manduca sexta</Emphasis> L.

This study examined the effects of dietary casein and sucrose levels on nutrient intake, and distinguished the effects of carbohydrate and protein consumption on growth, fat content, pyruvate metabolism and blood trehalose level of 5th instar Manduca sexta larvae. Growth increased with increasing casein consumption but was unaffected by carbohydrate intake. Fat content also increased with carbohydrate consumption, but on carbohydrate-free diets fat content increased with increased protein consumption. Blood trehalose level and pyruvate metabolism were examined by nuclear magnetic resonance spectroscopy analysis of blood following administration of (3-(13)C)pyruvate. On diets containing sucrose, blood trehalose increased with increasing carbohydrate intake, and on most diets trehalose was synthesized entirely from dietary sucrose. Pyruvate cycling, indicated by the alanine C2/C3 (13)C enrichment ratio, increased with carbohydrate consumption reflecting increased glycolysis, and pyruvate decarboxylation exceeded carboxylation on all sucrose diets. Larvae that consumed <75 mg/day sucrose were gluconeogenic, based on the [2 (trehalose C6)(Glx C3/C2)]/alanine C2] (13)C enrichment ratio. On carbohydrate-free diets, blood trehalose levels were low and maintained entirely by gluconeogenesis. Blood trehalose level increased with increasing protein intake. Pyruvate cycling was very low, although many insects displayed higher levels of pyruvate decarboxylation than carboxylation. All gluconeogenic larvae displayed alanine (13)C enrichment ratios <0.35 and had blood trehalose levels <50 mM.     

Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution

Devitrification has been determined to be one of the major causes of cell death in cryopreservation by vitrification method. Reliable quantification of the nucleation and growth of ice crystals of devitrification is of great importance for the optimization of the vitrification solutions. In the present study, cryomicroscopy was used to investigate the nucleation and growth of ice crystals in concentrated glycerol aqueous solution (60 wt%) in the presence of sucrose, trehalose, maltose and lactose. Results showed that sucrose rather than trehalose seems to be the most effective one to inhibit the nucleation and ice growth, despite the excellent inhibitory ability of trehalose on ice growth that has been confirmed in many researches. Hence, for ice inhibition, sucrose was a more effective disaccharide additive to suppress nucleation and growth of ice crystals that occurred during devitrification in concentrated glycerol solutions.     

Infectivity of beetle spiroplasmas for new host species

Five beetle spiroplasmas, the Colorado potato beetlespiroplasma (CPBS, strain LD-1), the Cantharis carolinusspiroplasma (CCBS, strain CC-1), the Ellychnia corrusca fireflyspiroplasma (FS, strain EC-1), the Diabrotica undecimpunctatacorn rootworm spiroplasma (CRS, strain DU-1), and the Spiroplasmafloricola fall flower spiroplasma (FFS), all associated withbeetles, were fed to beetles (Maladera matrida and Carpophilushumeralis) and mosquitoes (Aedes aegypti and Culex pipiens). CPBSand CCBS were also injected into M. matrida. Attempts to recoverspiroplasmas from regurgitates and hemolymph were conducted 1–10days after their introduction. After day 1, orally administeredspiroplasmas could not be recovered from M. matrida beetles;however, at 2–5 days, four out of five spiroplasmas wererecovered from adult C. humeralis. Injected spiroplasmas survivedin the hemolymph of M. matrida beetles for a relatively longperiod (at least 22 days). All five spiroplasmas were recoveredfrom mosquitoes 1 day post feeding, but only two (CCBS and CRS)survived for five or more days. The results show short andvariable persistence in orally challenged non-host insects, withgeneral failure to pass the gut barrier. Such evidence should beconsidered when attempting to use these microbes in biocontrolprograms.     

Nutritional Requirements of the Nematophagous Fungus Hirsutella rhossiliensis

Six natural media were examined for growth and sporulation of six isolates of the nematophagous fungus Hirsutella rhossiliensis , using solid and/or liquid culture. Twenty carbohydrates, 19 nitrogen (N) compounds, and nine vitamins were also tested for their effects on growth, sporulation, and spore germination of a further three isolates (ATCC46487, OWVT-1 and JA16-1). Variations in nutritional requirements existed among the fungal isolates. In general, V-8 juice agar (VA), cornmeal agar and potato dextrose agar were good media for growth, and malt extract agar, VA and yeast dextrose agar were good for sporulation of all six isolates. Glycogen was the best and sucrose, inulin, D- ( + ) - trehalose and soluble starch were also good carbon (C) sources for growth and spore germination of the three isolates ATCC46487, OWVT-1 and JA16-1 in both liquid and solid culture. None of the isolates utilized D- ( + )xylose as a C source. L- sorbose, D- ribose, citric acid and D- fructose were poor for growth of all isolates. The best C source for sporulation was D- ( + )-trehalose for ATCC46487, D- sorbitol for OWVT-1 and D- ( + )-cellobiose for JA16-1. Casein was the best N source for growth of ATCC46487 and OWVT-1, while peptone was best for JA16-1. L- asparagine, L- proline, and peptone were also good for growth of all three isolates. L - cystine was not utilized by H. rhossiliensis and DL- methionine inhibited growth of all isolates. Spore germination of all isolates was well supported by most N compounds examined but was inhibited by L- cystine. No significant difference in sporulation of ATCC46487 was observed among the N sources. DL- threonine was the best N source for spore production by OWVT-1 and L- phenylalanine was best for JA16-1. Vitamins generally enhanced fungal growth and sporulation, with thiamine having the greatest influence. Excluding some vitamins individually from the medium containing all other test vitamins sometimes increased growth and/or sporulation of certain isolates.     

Evidence for a nonrandom base sequence in a Bacillus pumilus plasmid: EcoR1 endonuclease digestion of pPL576.

EcoR1 endonuclease digested the Bacillus pumilus plasmid pPL576 (molecular weight similar to 28 X 10-6) into three distinct size classes of linear fragments. The molecular weights of the fragments are 13.0 X 10-6, 0.5 X 10-6, and 6.5 X 10-6 by sucrose gradient analysis. By electron microscope analysis the three fragments account for about 99% of the intact plasmid. These results indicate that pPL576 molecules contain a nonrandom base sequence, and are consistent with the interpretation that pPL576 is autonomous and not the result of cyclization of random chromosome fragments.     

Fusion-mediated transfer of plasmids into Spiroplasma floricola cells.

We have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells.     

Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti

Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in S. meliloti and R. leguminosarum also acted as very effective competitors of [14C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for S. meliloti and R. leguminosarum did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S. meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed.