A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl₄) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl₄ dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spe I fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt⁻) phenotype. One cosmid (pT31) complemented the Pdt⁻ phenotype of CTN1 and conferred CCl₄ transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.     

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.     

Study of PGM mutation mechanism in Yersinia pestis (plague pathogen) vaccine strain EV76

Yersinia pestis vaccine strain EV76 is a mutant of the virulent strain which has lost the pigmentation phenotype (Pgm+). This phenotype includes three characteristics: it absorbs pigments from agar media (Hms+), produces a siderophore yersiniabactin (Ybt+), and causes a lethal disease after subcutaneous inoculation of laboratory animals (Vir+). These characteristics are lost simultaneously after high frequency spontaneous deletion of 10 kB fragment of chromosomal DNA, termed the pgm locus. We compared the pgm locus-associated genetic and phenotypical properties of the vaccine strain with those of a typical Pgm- deletion mutant of a virulent strain. The results indicate that Pgm- phenotype of the vaccine strain results not from the deletion of the pgm locus, but from the insertion inactivation of the genes located in this locus. In contrast to the deletion mutant, the vaccine strain carries sequences detected by hybridization and PCR, which are complementary to the pgm locus genes. Moreover, the vaccine strain differed from the deletion mutant by a low level of Hms+ expression, a slower rate of cell death under iron-chelated conditions at 37 degrees C, "residual virulence" upon subcutaneous inoculation, and capacity to form revertants which restore the characteristics of Pgm+ phenotype after cell growth at 12 degrees C.     

Coat colour inheritance in Soay, Orkney and Shetland sheep

Analysis of the numerical proportions of Soay, Orkney and Shetland sheep of different colours together with test matings, produced results compatible with the hypothesis that these breeds have a multiple allelic series at locus A , white ( A ₁) being dominant to grey ( A ₂) and both being dominant to the gene for self-colour ( A ₅). The alleles at the A locus are epistatic to the alleles for pigment production at locus B , black ( B ₁) being dominant to brown ( B ₂).     

Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains

Abstract Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf -β₃ null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf -β₃ null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α₅- and β₁-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β₃ or neutralizing antibodies against fibronectin or the α₅-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf -β₃ null mutants; the importance of TGF-β₃ to restore their normal pattern of expression; and the crucial role of fibronectin and the α₅-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf -β₃ null mutant mice.     

Survival strategies of plasmid-carrier and plasmidless Escherichia coli strains under illuminated and non-illuminated conditions, in a fresh water ecosystem

A Comparative study, in illuminated and non-illuminated systems, was made to determine the survival strategies of plasmid-carrier and plasmidless bacteria in sterile river water. Two strains of Escherichia coli from river water were selected: one plasmidless, EC1, and one antibiotic-resistant strain, EC7, which showed plasmid bands. By matings with EC7 as donor and E. coli K12 strain J62 as recipient, transconjugants were generated, the J62₇ strain, which showed both antibiotic resistance and plasmid bands. Ethidium bromide curing of the EC7 strain generated the EC7₂ strain which showed a partial loss of resistance and a reorganization of plasmid bands. Under non-illuminated conditions the total number of cells detected by direct count and the number of culturable cells (injured and non-injured cells) remained practically constant throughout the period of incubation. In the illuminated systems, however, the number of cfu decreased in four of the five strains studied. The greatest decreases are those of the J62 strain, followed by those of the J62₇, EC1, EC7₂ and EC7 strains. Differences in survival strategies as a consequence of the presence or absence of plasmids are discussed.     

EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF_U, which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF _U does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF_U-promoted actin pedestal formation. EHECΔ espF _U efficiently colonized the rabbit intestine during co-infection with wild-type EHEC, but co-infection studies on cultured cells suggested that EspF_U produced by wild-type bacteria might have rescued the mutant. Significantly, EHECΔ espF _U by itself was fully capable of establishing colonization at 2 days post inoculation but unlike wild type, failed to expand in numbers in the caecum and colon by 7 days. In the gnotobiotic piglet model, an espF _U deletion mutant appeared to generate actin pedestals with lower efficiency than wild type. Furthermore, aggregates of the mutant occupied a significantly smaller area of the intestinal epithelial surface than those of the wild type. Together, these findings suggest that, after initial EHEC colonization of the intestinal surface, EspF_U may stabilize bacterial association with the epithelial cytoskeleton and promote expansion beyond initial sites of infection.     

Chlorate resistant mutants of Pseudomonas stutzeri affected in respiratory and assimilatory nitrate utilization and expression of cytochrome cd1

Abstract Chlorate-resistant mutants were generated by random insertion of the transposon Tn5 into genomic DNA of Pseudomonas stutzeri ZoBell strain and selected for loss of nitrate respiration (Nar phenotype). The mutants were differentiated by restriction-fragment analysis, by assaying for nitrate assimilation and for molybdenum co-factor activity, and by the amount of respiratory nitrate reductase. Two mutants, lacking both nitrate respiration and nitrate assimilation, over-produced an inactive nitrate reductase but synthesized in the presence of nitrate only a reduced amount of respiratory nitrite reductase (cytochrome cd ₁). Expression of cytochrome cd ₁ in these mutants was specifically induced by nitrate, suggesting a sensor system for this substrate.     

Cloning and sequencing of mutantpsbB genes of the cyanobacteriumSynechocystis PCC 6803

Ten strains from a collection of mutants ofSynechocystis 6803 defective in Photosystem II (PS II) function were transformed with chromosomal DNA of wild-type and mutant cells. Cross hybridization data allowed to identify four groups of PS II-mutants. Highly efficient transformation was observed between different mutant groups, but not within the groups. Restoration of photosynthetic activity of the mutant cells was also achieved by transformation with different parts of a 5.6 kbBam HI fragment of wild typeSynechocystis DNA containing thepsbB gene. Each group of mutants was transformed to photoautotrophic growth by specific subfragments of thepsbB gene. DNA fragments of four selected mutant strains hybridizing with thepsbB gene were isolated and sequenced. The mutations were identified as a single nucleotide insertion or substitution leading to stop codon formation in two of the mutants, as a deletion of 12 nucleotides, or as a nucleotide substitution resulting in an amino acid substitution in the other two mutants. Deletion of 12 nucleotides in mutant strain PMB1 and stop codon formation in strain NF16 affect membrane-spanning regions of the gene product, the CP 47 protein.     

Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes . The Ku genes, Ku70 and Ku80 , encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes , the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80 . The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens -mediated genetic transformation. The average homologous recombination frequency was 73.3 ± 25.2% for the areA/nit-2 -like nitrogen regulatory gene ( tnr ) in Ku80⁻ mutants, about 33-fold higher than that in wild-type controls. A high frequency ( c . 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80 ⁻ mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes , providing valuable information on the basic mechanisms of host invasion.     

Copper acquisition by the SenC protein regulates aerobic respiration in Pseudomonas aeruginosa PAO1

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage ( attP _TG1 ) and bacterial ( attB _TG1 ) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis . The attB _TG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N -succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis , however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB _TG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus . The gene encoding the putative TG1 integrase ( int _TG1 ) was located adjacent to the attP _TG1 site. TG1 integrase deduced from the int _TG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli .     

Chromosomal Basis of the Merozygosity in a Partially Diploid Mutant of Pneumococcus

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.