Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.     

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.     

Evaluation of the efficacy of zwitterionic dodecyl carboxybetaine surfactants for the extraction and the separation of mycoplasma membrane protein antigens

The ability to extract mycoplasma membrane protein antigens using the alkyl carboxybetaine surfactants (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM) and (N-dodecyl-N,N-dimethylammonio)undecanoate (DDMAU, CMC = 0.13 mM) was assessed by protein titration and SDS-PAGE analysis. The maximum yields of membrane protein solubilization ranged from 20 to 90%, depending upon both the mycoplasma membrane investigated and the surfactant used. In five of six cases, the extraction was optimal for surfactant concentrations of ca. 25 mM. DDMAB displayed a higher efficiency in membrane protein extraction. The order of efficiency for both surfactants was Spiroplasma melliferum > Acholaplasma laidlawii > Mycoplasma gallisepticum. In contrast, DDMAU proved much more selective. The order of selectivity was M. gallisepticum > S. melliferum > A. laidlawii. The highest selectivity was recorded for the major proteins p67 and spiralin of M. gallisepticum and S. melliferum, respectively. For p67, notably, DDMAU proved superior to 10 other surfactants. Dot immunobinding and crossed immunoelectrophoresis analyses showed that both dodecyl carboxybetaines were suitable as membrane protein-solubilizing agents in immunological techniques. Furthermore, these surfactants did not exhibit effects adverse to the activity of A. laidlawii membrane NADH oxidase. One promising application of DDMAU is the separation of membrane proteins by ion-exchange HPLC as illustrated by the good resolution of M. gallisepticum membrane proteins and purification of p67 to almost homogeneity. These data show that dodecyl carboxybetaine surfactants are useful for the extraction of mycoplasma membrane antigens under mild conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of coronary microvascular collaterals to activation of ATP-sensitive K+ channels

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317-1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat-beta-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.     

The Torsion-Inversion-Bending Energy Levels in the S1(n, pi*) Electronic State of Acetaldehyde

The membrane permeabilisation properties of six linear natural 18-residue peptaibols, termed trichorzins PA, have been assessed on liposomes and on mollicutes (trivial name, mycoplasmas), a class of parasitic bacteria characterized by a small genome, the lack of a cell wall, a minute cell size, and the incorporation in their plasma membrane of exogenously supplied cholesterol. The trichorzins PA used in this study (PA II, PA IV-VI, PA VIII, and PA IX) differ between them by amino acid or amino alcohol substitutions at positions 4, 7, and 18, and form slightly amphipathic alpha-helices. They proved bactericidal for mollicutes belonging to the genera Acholeplasma, Mycoplasma, and Spiroplasma, with minimal inhibitory concentrations (3.12相似文献   

Isolation and characteristics of UV-resistant revertants of recA-strains of Escherichia coli K-12

The paper presents the results of a study of UV-resistant revertants of some recA strains induced by different mutagens. It is shown that some of produced revertants differ from original recA strains in some properties. It is established that all UV-resistant revertant fall into three phenotypic groups on their recombination proficiency in crosses with different donor strains (Hfr C, Hfr 3.0SO, F' W1655) and in their sensitivity to UV and gamma-rays. It is concluded that all UVr revertants are rec A+ and carried mutations either in the same recA gene (true reversion or intragenic suppression) or in genes closely linked with recA.     

Cloning, sequencing, and characterization of the recA gene from Rhodopseudomonas viridis and construction of a recA strain

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec- phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec- phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.     

Construction of recombination-deficient strains of Streptococcus gordonii by disruption of the recA gene

Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.     

Spiroplasma platyhelix sp. nov., a new mollicute with unusual morphology and genome size from the dragonfly Pachydiplax longipennis

Spiroplasma strain PALS-1T from the gut of the dragonfly Pachydiplax longipennis was shown to be distinct from other species, groups, and subgroups of the genus Spiroplasma as determined by reciprocal serological metabolism inhibition and deformation tests. However, this strain cross-reacted extensively with representatives of other groups when it was used as an antigen. Electron microscopy of cells of strain PALS-1T revealed cells surrounded by a single cytoplasmic membrane. Light microscopy revealed helical cells that exhibited twisting motility rather than rotatory or flexing motility. Variations in the tightness of coiling were transmitted from one end of the helix to the other. The strain was resistant to penicillin, which confirmed that no cell wall was present. The organism grew well in M1D and SP-4 liquid media under either aerobic or anaerobic conditions. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. The optimum temperature for growth was 30 degrees C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32 degrees C. Strain PALS-1T catabolized glucose and hydrolyzed arginine but not urea. The guanine-plus-cytosine content of the DNA was 29 +/- 1 mol%. The genome size was 780 kbp, the smallest genome size in the genus Spiroplasma. Strain PALS-1 (= ATCC 51748) is designated the type strain of a new species, Spiroplasma platyhelix.     

Spiroplasma lineolae sp. nov., from the horsefly Tabanus lineola (Diptera: Tabanidae)

Spiroplasma strain TALS-2T from the viscera of the striped horsefly, Tabanus lineola, collected in Georgia was serologically distinct from other Spiroplasma species, groups, putative groups, and subgroups. Light and electron microscopy of cells of strain TALS-2T revealed helical motile cells surrounded only by a single cytoplasmic membrane. The organism grew in M1D and SP-4 liquid media. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. Growth in liquid media was serum dependent. The strain passed through 220-nm filter pores, but was retained in filters with 100-nm pores. The optimum temperature for growth was 30 degrees C. Multiplication occurred at temperatures from 20 to 37 degrees C, with a doubling time at the optimum temperature of 5.6 h in M1D broth. Strain TALS-2T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was 25 +/- 1 mol%. The genome size was 1,390 kbp. Six isolates serologically similar to strain TALS-2T were obtained from the same host in coastal Georgia. Three strains closely related to strain TALS-2T were isolated from the horsefly Poeciloderas quadripunctatus in Costa Rica. Strain TALS-2T (= ATCC 51749), a representative of group XXVII, is designated the type strain of a new species, Spiroplasma lineolae (Mollicutes: Entomoplasmatales).     

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.     

Colicin U, a novel colicin produced by Shigella boydii

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1.