Genome analysis of a Mycoplasma hyorhinis strain derived from a primary human melanoma cell line

The complete genome of Mycoplasma hyorhinis strain MCLD has been sequenced and annotated. This genome differs by the inversion of a 14.4-kb and a 3.7-kb fragment and the deletion of a 9.9-kb fragment from M. hyorhinis strain HUB-1, isolated from swine respiratory tract. The genome revealed 778 coding sequences (CDSs), with a limited number of vlp genes encoding variable surface lipoproteins.     

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.     

Monoclonal antibodies to Mycoplasma hyorhinis surface antigens: tools for analyzing mycoplasma-lymphoid cell interactions

A library has been constructed of approximately 50 monoclonal antibodies that recognize antigens of Mycoplasma hyorhinis. Characteristics of six antibodies are discussed. Each reacts with a discrete determinant borne on a protein-containing molecule of distinct molecular size. Three of these respective antigens are expressed at the surface of mycoplasmas colonizing lymphoblastoid cells in culture. Of these three surface antigens, two are selectively expressed on strain GDL but not on strain BTS-7 of M. hyorhinis, thus defining strain-restricted immunological specificities within these species. One monoclonal antibody of the IgM class (mu, kappa) mediated marked complement-dependent growth inhibition of M. hyorhinis in broth culture. These monoclonal reagents should facilitate analysis of mycoplasma surface architecture, and the molecular interactions of these organisms with the host cell surface.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

芽孢杆菌SK007的分类鉴定及其拮抗植物病原菌的功能分析

【背景】农业生产中,发掘和利用具有生防功能的微生物资源是保障粮食安全和提高作物产量的重要举措。【目的】明确土壤中芽孢杆菌SK007的分类地位,验证其对多种植物病原菌的拮抗作用,挖掘潜在的生防功能。【方法】通过16SrRNA基因和基因组分析方法确定分离菌株SK007的分类地位;采用平板对峙法研究该菌株对番茄灰霉病菌、白菜黑斑病菌、烟草赤星病菌、小麦赤霉病菌、马铃薯干腐病菌等植物病原菌的拮抗作用;采用AntiSMASH分析和预测菌株SK007的抗生素相关基因。【结果】基于16SrRNA基因、全基因组序列、平均核苷酸一致性和DNA同源性分析,结果表明菌株SK007属于Bacillus velezensis,并且具有产生脂肽类抗生素和聚酮类抗生素的基因,对多种植物病原真菌有较强的抗性。此外,菌株SK007基因组中抗生素基因簇数目较多,丰富度高。【结论】芽孢杆菌SK007在拮抗植物病原菌方面有许多优良性状,具有促进作物抗病和增产的潜力。     

Potential Use of Surface-active Agents for Controlling Mycoplasma Contamination in Animal Cell Cultures

Seven nonionic detergents, which were determined to be relatively nontoxic to selected animal cell cultures, were tested for their lethal effect on the GDL strain of Mycoplasma hyorhinis. Of the seven detergents tested, five were found to cause complete lysis of the organism in vitro within 24 hr at 37 C. These detergents included Triton WR-1339 and Tweens 20, 40, 60, and 80. When different concentrations of the detergents were tested, Tween 80 was found to be the most effective and Triton WR-1339 the least effective in lysing the mycoplasmata. These same five detergents were used to treat a rat nephroma cell line which was chronically infected with the GDL strain. The mycoplasmata were eliminated from those cultures treated with Triton but they persisted in cultures exposed to the Tween compounds. The Triton-treated cells remained free from infection over a 7-month period, as determined both by cultural methods and fluorescent-antibody staining. The "cure" was effected by treating the cells for either 48 hr with maintenance media containing 1 mg of Triton per ml or for 96 hr with a concentration of 500 mug/ml. Triton was also effective in eliminating the GDL, strain from experimentally infected rat embryo cells after a 48-hr treatment with a concentration of 1 mg/ml. Four other species of Mycoplasma, which were completely lysed by Triton in vitro, were not eliminated from experimentally infected cells by a single treatment with Triton, although the severity of the infection was apparently reduced.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Genes, encoding invasion factors in Listeria monocytogenes strains isolated in the European part and the far east of Russia

The collection of 76 Listeria monocytogenes strains isolated from humans, animals and food products was screened with PCR to reveal genes, which encode invasion factors of the internalin family. Obtained results demonstrated the correlation between the strain specific polymorphism of the revealed internalin genes and the source of the strain.     

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.     

传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达

核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %～ 98 6 %,氨基酸的同一性为 91 0 %～ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4 5kD表达蛋白     

Genetic transformation of genes for protein II in Neisseria gonorrhoeae.

The protein II (PII) outer membrane proteins of Neisseria gonorrhoeae are a family of heat-modifiable proteins that are subject to phase variation, in which the synthesis of different PII species is turned on and off at a high frequency. Transformation of PII genes from a donor gonococcal strain into a recipient strain was detected with monoclonal antibodies specific for the PII proteins of the donor. Individual PII protein-expressing transformants generally bound only one donor-specific PII monoclonal antibody. Recovery of transformants expressing a donor-specific PII protein depended on the PII protein expression state of the donor: the transformed population bound only monoclonal antibodies specific for PII proteins that were expressed in the donor. Colony variants with an altered frequency of switching of PII protein expression were isolated, but the altered switch phenotype did not cotransform with the PII structural gene. These results provide genetic evidence that PII proteins are the products of different genes and that expressed and unexpressed forms of the PII gene are different from each other.     

Exotoxin A of Pseudomonas aeruginosa: evidence that Domain I functions In receptor binding

We have constructed defined deletions in the structural gene of Pseudomonas aeruginosa exotoxin A (ETA) In order to probe the function of Domain t of this protein. Three forms of the gene containing specific deletions were expressed in a strain of Escherichia coli K12 with lesions in the htpR and ion genes; extracts containing the gene products were tested for ADP-ribosylation activity, cytotoxicity, and ability to protect sensitive cells from the cytotoxic action of authentic ETA. Two of the mutant ETAs gave concentration-dependent protection against authentic ETA, and protection correlated with the presence of the bulk of Domain I. The results support the notion that Domain I functions in binding the toxin to specific cell-surface receptors.