Temperature Ranges,Growth Optima,and Growth Rates of Spiroplasma (Spiroplasmataceae,class Mollicutes) Species

A new method was developed for determination of the doubling times of spiroplasmas. In this procedure, the time required for medium acidification of tubes in tenfold dilution series was recorded. Sixty-four spiroplasma strains, representing 24 groups and 11 subgroups, were studied. Eight strains representing putative new groups were also included in the study. Doubling times at 5, 10, 15, 20, 25, 30, 32, 37, 41, and 43°C were determined. The range of temperatures for spiroplasma growth was 5°–41°C. Twenty-three spiroplasmas had optima of 30°C, 29 had optima of 32°C, and 13 had optima of 37°C. The fastest growing spiroplasma was the MQ-4 strain (group XI), with a doubling time at optimal temperature of 0.6 h. The slowest was the Jamaican corn stunt strain B655 (subgroup I-3), with an optimal doubling time of 36.7 h. Spiroplasma strain B31 (group IV) had the widest range (5°–41°C), while the DW-1 strain and some subgroup I-3 strains had the narrowest, growing only at 25° and 30°C. Some spiroplasmas grew well at 41°C, but none grew at 43°C. The ability of spiroplasmas to withstand a wide range of temperatures may reflect the conditions to which they are exposed in nature, including the temperatures of the insect, tick, and/or plant hosts in which they are carried and the plant surfaces from which they may be acquired by arthropods.     

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.     

Proposed subgroups of spiroplasmas of high guanine plus cytosine content, group IV

The plant surface and insect-inhabiting spiroplasmas of group IV, unlike other spiroplasmas, have not been demonstrated to utilize arginine. They require cholesterol for growth, produce spots and films on some media, and do not hydrolize arbutin. Electrophoretic and serological comparisons of strains from North America and Europe indicate the existence of strain differences within group IV. This study provides evidence for the existence of three discrete subgroups, group IV-(1) represented by temperate American strains, group IV-(2) represented by subtropical American strain PPS1, and group IV-(3) represented by Mediterranean and French strains.     

Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family. Received: 23 April 1997 / Accepted: 31 May 1997     

Differentiation of group VIII Spiroplasma strains with sequences of the 16S-23S rDNA intergenic spacer region

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S-23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S-23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.     

<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

Identification of New World Leishmania species from Peru by biochemical techniques and multiplex PCR assay

We have characterized diverse strains or species of Leishmania isolated in humans that are currently circulating throughout Peru, by means of isoenzymatic characterization, kDNA analysis by restriction enzymes, and multiplex PCR assay. The cluster analysis gave five groups. Cluster 1 includes L. (L.) donovani together with the isolates LP4 and LP7, forming the donovani complex. Thus, this complex corresponds to the New World visceral form, L. (L.) chagasi. Cluster 2 is formed by the isolates LP1-LP3, LP6, LP10, LP9, and LP11, phylogenetically intermediate between Cluster 1 and Cluster 3, or they can be treated as hybrids. Cluster 3 is divided into two subgroups: one formed by L. (V.) peruviana, together with the isolates LP14 and LP5, and the second one formed by L. (V.) brazilensis and the isolate LP8. These two subgroups form part of the brazilensis complex. The three strains of L. (L.) infantum [L. (L.) infantum I and II and la LSI] make up Cluster 4. In Cluster 5, we include the three Mexican strains (LM1-LM3) forming one subgroup while we would place L. (L.) amazonensis in another subgroup. These two subgroups would comprise the complex mexicana.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

How do Japanese isolates ofVerticillium dahliae correspond with standardized VCG testers?

We examined the vegetative compatibility of 56 Japanese isolates provisionally assigned to four subgroups ofV. dahliae to estimate the genetic relatedness with testers of the standardized VCGs. Subgroup J1 was assigned to VCG 2A/B as a new category of assignment. Subgroup J2, except isolate Vdt 110, was assigned to VCG 2A, and subgroup J3, except isolate Vdf 1, was assigned to VCG 2B. Isolates Vdf 1 and Vdt 110 were assigned to VCG 2A/B. Subgroup J4 was assigned to two subgroups, VCG 4B for Vde 1 and VCG 4A/B for FY 3 and HR 1. Four isolates were compatible with both VCG 2 and 4. Isolate U56 was compatible with VCG 2A/B and 4A. Isolates of VCG 2A, Vdt 9 and FF1, were compatible with either VCG 4A or 4A/B. One isolate of VCG 2B, Vdp-4, was compatible with VCG 4A. Three isolates of subgroup J2 showed weak reactions with the testers of VCG 4. These isolates may be “bridging strains”. Japanese isolates were composed of two VCGs, 2 and 4, “bridging strains” compatible with these VCGs, and some self-incopatible isolates. Testers of VCG 1 and VCG 3 did not show any reactions with the Japanese isolates.     

Relation of in vivo morphology to isolation of plant spiroplasmas

Two procedures were developed to isolate plant spiroplasmas directly onto DG-2 agar plates or in DG-2 broth without subcultures or dilutions. The frequency of successful spiroplasma isolations was increased by centrifuging samples, after passing through a 0.45-μm filter, at 25,000 × g for 1 h. Spiroplasmas were obtained from peach, cherry, Madagascar periwinkle, and celery with typical symptoms of the Green Valley strain of X disease (GVX), from peach with typical symptoms of the peach yellow leaf roll strain of X disease (PYLR), from Madagascar periwinkle with typical symptoms of aster yellows (AY), from celery with atypical symptoms of GVX (mild GVX), from plantago with atypical symptoms of aster yellows (mild AY), and from stubborn-diseased citrus. Isolations were consistent (>90%) from plants with mild GVX, mild AY, and citrus stubborn, while isolations were inconsistent (0–9%) from plants with typical symptoms of GVX, PYLR, and AY. The role of the isolated spiroplasmas in plant disease was not determined in this study. All spiroplasma isolates were serologically indistinguishable fromSpiroplasma citri. Spiroplasmas were seen in electron micrographs of 8 out of 9 examined plants from which spiroplasmas were isolated. However, electron micrographs of all 13 examined plants from which no spiroplasmas were isolated contained mycoplasma-like organisms (MLOs) but no, spiroplasmas. These results indicate that there is a correlation between helical MLOs in vivo and successful isolation of spiroplasmas, and that plants may be infected with bothS. citri and nonhelical mycoplasmas.     

Interspecific hybridizations in the Elymus semicostatus group (Poaceae).

Meiotic pairing in 16 interspecific hybrids in the genus Elymus is reported. The hybrids were made among seven species in the Elymus semicostatus group, viz., E. semicostatus, E. validus (subgroup I), E. abolinii (subgroup II), E. fedtschenkoi, E. nevskii, E. praeruptus (subgroup III), and E. panormitanus (subgroup IV). All species are tetraploid (2n = 4x = 28) and possess the SY genomes. Meiotic pairing was distinctly higher in hybrids made within subgroups than between subgroups, but the genomes in E. panormitanus have differentiated from those in the other species. These results generally support the subdivision of the E. semicostatus group based on morphological data but also indicate that the subgroups are more distantly related than previously believed, and that the group may be nonmonophyletic.     

黄土高原根瘤菌数值分类及DNA-DNA杂交

黄土高原位于我国内陆,气候比较干旱,生长的植被较少,水土流失严重,而有些豆科植物如锦鸡儿(Caragana sinica)、苦豆子(Sophora alopecuroides)、甘草(Glycyrrhiza uralensis)、苦马豆(Swainsoniasalsula)、洋槐(Robinia pseudoacacia)等却能很好生长.这些豆科植物的生长,在防风固沙、保持水土、绿化环境、作为饲用牧草等方面起着很重要的作用.但人们对于黄土高原野生豆科植物根瘤菌的研究尚很少.为此,作者在地处黄土高原的陕西、宁夏及甘肃的部分地区进行了广泛的根瘤菌资源调查.在此基础上,对分离的部分菌株进行了数值分类和DNA同源性分析.     

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.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water.

Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water

M. GENNARI AND F. DRAGOTTO. 1992. Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

Puffing patterns during the fourth larval instar in Chironomus pallidivittatus salivary glands

The puffing pattern of polytene chromosomes in salivary glands from Chironomus pallidivittatus larvae and prepupae has been studied in glutaraldehyde-acetic acid fixed, lactic acid flattened preparations. Some observations were also made on F1 hybrid species C. pallidivittatus X C. tentans. Concerning the situation of puffing in Balbiani rings (BR), 2.783 chromosomes IV from 188 animals were scored. In standard 4th instar larvae, BR2 appears expanded, BR3 smaller but not collapsed and BRI either reduced of collapsed. During the first days following the red-head stage, which signals the beginning of the 4th instar, larvae show a large BR1; later it reduces and tends to collapse. At the end of the 4th instar, prepupae again may present an expanded BR1. On the contrary, the size of BR2 and BR3 remains unchanged from the red-head stage to the prepupa. A variable accumulation of droplets has been observed to occur in BR2 and BR1 from dated larvae and prepupae.--A characteristic pattern of puffing was found in prepupae, which consisted in the appearance of conspicuous puffs at regions I-6B, I-7B, I-7B, I-18C, III-9B and IV-4B. Puffs at I-2B, I-3B, I-9A,I-11C,II-4A, and IV-4B were observed during most of the 4th larval instar, as well as in late larvae and prepupae. Among all these puffs, those at I-7B, I-9A, I-17B, and IV-4B frequently showed variable amounts of droplets.     

Phosphorylation and functions of inhibitor-2 family of proteins

Protein phosphatase-1 (PP1) is an essential protein Ser/Thr phosphatase that is extraordinarily conserved from yeast to human, and Inhibitor-2 (I-2) is the most ancient of the heat-stable proteins specific for PP1. We identified novel I-2 homologues in Caenorhabditis elegans (Ce) and Xenopus laevis (Xe) and compared them to the I-2 proteins from Homo sapiens (Hs), Saccharomyces cerevisiae (GLC8), and Drosophila melanogaster (Dm). The Ce I-2 and Dm I-2 showed the highest potency inhibition of rabbit PP1 with IC50 near 5 nM compared to Hs I-2 and Xe I-2 with IC50 between 10 and 50 nM and GLC8 with >100-fold lower activity. Inhibition of PP1 bound to Nek2 kinase activated the kinase to phosphorylate a C-Nap1 domain substrate. All the species of I-2 except GLC8 activated the Nek2::PP1 to the same extent as microcystin-LR. Only Hs I-2 and Xe I-2, not the I-2 proteins more divergent in sequence, directly activated human Aurora-A kinase. Various species of I-2 have a common PxTP phosphorylation site that showed a wide range of reactivity with GSK3, ERK, or CDC2/cyclinB1 kinases. The Suc1 subunit of CDC2/cyclinB1 enhanced reactivity with I-2, consistent with this being a site of mitotic phosphorylation. The results show species specificity among the I-2 family within the context of conserved PP1 inhibitory activity and variable phosphorylation by Pro-directed kinases.