Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: Determination of the minimal replicon and functionality identification of the putative sso

In order to determine the minimal replicon and the single strand origin (sso) of the plasmid pM4, different fragments of pM4 were amplified by polymerase chain reaction (PCR) and cloned into pBEm, a replication probe vector for Lactobacillus. The deletion analysis results showed that the minimal replicon of pM4 could be determined within a 1280 bp fragment consisting of double strand origin (dso) and rep gene encoding replication protein. Based on plasmid segregation stability assay and its ability to convert single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) by Southern hybridization, an sso of replication was located at nucleotides −118–92 in the plasmid pM4, about 300 bp upstream of dso. In addition, the host range assay indicated that plasmid pM4 could replicate in L. casei 05–21, L. rhamnosus AS 1.2466^T and L. plantarum 05–19 of all the tested Lactobacillus strains. Analysis of the pM4 replicon will allow its use in constructing a food-grade vector for application in food industry.     

Isolation and characterization of a temperature-sensitive conditional mutant of Escherichia coli altered for the control of phosphate-regulated proteins

We have identified a conditional mutation which confers a ple?otropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C. This mutation was located at minute 89.5 on the E. coli chromosome in a gene we have called cpr for conditional phosphate-regulated.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene

A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively. We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene. DNA replication in these mutants has a fast-stop phenotype, indicating that the S. aureus dnaC gene encodes the replicative DNA helicase required for the elongation step. These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair. The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S. aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects.     

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFVI and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFVI-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and AH. Comparison of the FR-I elements from these strains with that from pFVI revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFVI and pFZI, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5–3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.     

Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

Summary The ribosomal proteins of temperature-sensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis. Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins. The proteins altered in these mutants are S2, S4, S6, S7, S8, S10, S15, S16, S18, L1, L3, L6, L10, L11, L14, L15, L17, L18, L19, L22, L23 and L24. A large number of them (25 mutants) have mutations in protein S4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit. The importance of these mutants for structural and functional analyses of ribosomes is discussed.     

Isolation of the gene coding for Caenorhabditis elegans Rac2 homologue, a Ras-related small GTP-binding protein

When screening a Caenorhabditis elegans genomic library using the human Racl cDNA as probe, a hybridizing fragment of 2.7 kb was isolated which contained four exons with high sequence similarity to CeRacl, coding for the nematode homologue of the Ras-related small GTP-binding protein Racl. The putative translational product of 195 amino acids (aa) from the exons displayed 88% identity to the sequence of CeRacl. Interestingly, three alterations were found in the N-terminal ‘effector domain’ (residues 22–45) which hitherto was identical among all known Rac p21s, suggesting that CeRac2 might have different targets/functions for nematode development. Additionally, an insertion of 4 as was found in the hypervariable region at the C terminus of CeRac2.     

Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59–299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis.     

Sequencing and modification of psbB,the gene encoding the CP-47 protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

一株猪葡萄球菌的分离鉴定、生物学特性研究及全基因组测序分析

【背景】2021年6月,广东省茂名市某散养户送检了一头发病仔猪,猪身上长有脓疱,四肢关节肿大,关节内可见脓液。【目的】确定引起仔猪发病的病原菌,分析其药物敏感性,为临床用药提供指导;对分离菌株进行全基因组序列分析,挖掘其毒力因子和耐药基因,揭示该菌致病和耐药的分子机制。【方法】取关节脓液分离细菌;通过革兰氏染色、16S rRNA基因和全基因组测序分析,鉴定细菌种类;通过溶血试验、血浆凝固酶试验和生长曲线测定,确定分离菌株的溶血活性、血浆凝固酶活性和生长特性;用小鼠感染模型评估分离菌株的致病性;用纸片扩散法测定分离菌株的药物敏感性;通过全基因组序列分析挖掘分离菌株的毒力因子和耐药基因。【结果】分离菌株被鉴定为猪葡萄球菌(Staphylococcus hyicus);该菌不溶血,无血浆凝固酶活性,在胰蛋白胨大豆肉汤培养基中于37℃、120r/min条件下生长良好;小鼠感染试验结果显示,该菌具有高致病性;药敏试验结果显示,该菌对苯唑西林、大观霉素等7种药物敏感,对青霉素G、红霉素等9种药物耐药;全基因组序列分析结果显示,该菌携带多个毒力因子和耐药基因。【结论】从发病仔猪的关节脓液中分离到一株猪葡萄球菌,可用苯唑西林、大观霉素等药物防控该菌感染;解析了该菌的基因组信息,为后续深入研究该菌致病和耐药的分子机制奠定了基础。     

Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid

Summary The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning. In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene. The purity of the final preparation was more than 95%, as determined by densitometry. The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene. Therefore, it was concluded that the protein was the sopB gene product. Using anti-SopB serum, the SopB protein was detected in the cell lysates of F⁺, F, and Hfr strains. The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322.     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein RepA

Summary In vitro mutagenesis with hydroxylamine of a ParD^- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42° C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3 end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.     

Generation and characterization of a conditional deletion allele for Lmna in mice

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4–8 postnatal (P) weeks or P16–18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna^flox/flox mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna^Δ/Δ) generated by germline Cre expression display postnatal lethality at P16–18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.