Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

新城疫病毒ZJ1株F蛋白裂解位点突变对其融合活性的影响

新城疫病毒ZJ1毒株是近年来在我国水禽中流行并能引起水禽严重发病和死亡的强毒株,其F蛋白裂解位点有多个碱性氨基酸分布。将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸,构建了重组表达质粒pCI-FT。分别将突变前后的F蛋白与该毒株的HN蛋白在COS-1细胞共表达,表明突变前后的F蛋白均有融合活性;分别将突变前后的F蛋白与该毒株的HN蛋白在CEF细胞共表达,表明突变后F蛋白被裂解的活性大大降低。以上研究为下一步在全长cDNA克隆水平上对F蛋白裂解位点氨基酸序列进行相应突变,研究毒力相关因素以及构建毒力致弱疫苗株等奠定基础。     

Characterization of a Bacillus cereus protease mutant defective in an early stage of spore germination.

Temperature-sensitive sporulation mutants of Bacillus cereus were screened for intracellular protease activity that was more heat labile than that of the parental strain. One mutant grew as well as the wild type at 30 and 37 degrees C but sporulated poorly at 37 degrees C in an enriched or minimal medium. These spores germinated very slowly in response to alanine plus adenosine or calcium dipicolinate. During germination, spores produced by the mutant rapidly became heat sensitive, but released dipicolonic acid and mucopeptide fragments more slowly than the wild type and decreased only partially in density while remaining phase white (semirefractile). In freeze-etch electron micrographs, the mature spores were deficient in the outer cross-patched coat layer. During germination, the spore coat changes associated with wild-type germination occurred very slowly in this mutant. Although the original mutant was also a pyrimidine auxotroph, reversion to prototrophy did not alter any of the phenotypic properties discussed. Selection of revertants that germinated rapidly or sporulated well at 37 degrees C, however, resulted in restoratin of all wild-type properties (exclusive of the pyrimidine requirement) including heat-stable protease activity. The reversion frequency was consistent with an initial point mutation, indicating that a protease alteration resulted in production of spores defective in a very early stage of germination.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Biosynthesis of L-DOPA by Aspergillus oryzae

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp/x 40.00+/-0.01 d mg/mg with parent and 182.86+/-0.02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q(p), there was significant enhancement (p < 0.0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment of Parkinson's disease and myocardium following neurogenic injury.     

A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol

Francisella tularensis is an intracellular bacterial pathogen, and is a category A bioterrorism agent. Within quiescent human macrophages, the F. tularensis pathogenicity island (FPI) is essential for bacterial growth within quiescent macrophages. The F. tularensis-containing phagosome matures to a late endosome-like stage that does not fuse to lysosomes for 1-8 h, followed by gradual bacterial escape into the macrophage cytosol. Here we show that the FPI protein IglD is essential for intracellular replication in primary human monocyte-derived macrophages (hMDMs). While the parental strain replicates robustly in pulmonary, hepatic and splenic tissues of BALB/c mice associated with severe immunopathologies, the isogenic iglD mutant is severely defective. Within hMDMs, the iglD mutant-containing phagosomes mature to either a late endosome-like phagosome, similar to the parental strain, or to a phagolysosome, similar to phagosomes harbouring the iglC mutant control. Despite heterogeneity and alterations in phagosome biogenesis, the iglD mutant bacteria escape into the cytosol faster than the parental strain within hMDMs and pulmonary cells of BALB/c mice. Co-infections of hMDMs with the wild-type strain and the iglD mutant, or super-infection of iglD mutant-infected hMDMs with the wild-type strain show that the mutant strain replicates robustly within the cytosol of hMDMs coinhabited by the wild strain. However, when the wild-type strain-infected hMDMs are super-infected by the iglD mutant, the mutant fails to replicate in the cytosol of communal macrophages. This is the first demonstration of a F. tularensis novel protein essential for proliferation in the macrophage cytosol. Our data indicate that F. tularensis transduces signals to the macrophage cytosol to remodel it into a proliferative niche, and IglD is essential for transduction of these signals.     

Isolation of an aggregation-less mutant of Dictyostelium discoideum with the expression of contact site A glycoprotein

We isolated mutants defective in aggregation (aggregation-less) by mutagenizing the "double-bypass" mutant HG592 of Dictyostelium discoideum as the parental strain. One of the mutants expressed the contact site A glycoprotein with an apparent molecular weight of 80 X 10(3) on the cell surface in the normal developmental stage and retained EDTA-stable cell contact as well as EDTA-sensitive cell contact. However, the mutant failed to aggregate on agar plates with bacteria. This mutant was designated HG700. We could not identify any differences between this mutant and the parental strain in levels of adenylate cyclase or extracellular phosphodiesterase activity, or in its chemotaxis toward cAMP. The mutant had greatly decreased the incorporation of [35S] sulfate into the particulate fractions of the cells starved for 6 h. This suggests that the modification by sulfation may crucially affect the mechanism of cell aggregation.     

A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus

A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5' terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of S. aureus.     

Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole

To identify host proteins involved in Legionella pneumophila intracellular replication, the soil amoeba Dictyostelium discoideum was analysed. The absence of the amoebal RtoA protein is demonstrated here to depress L. pneumophila intracellular growth. Uptake of L. pneumophila into a D. discoideum rtoA(-) strain was marginally defective, but this effect was not sufficient to account for the defective intracellular growth of L. pneumophila. The rtoA mutant was also more resistant to high-multiplicity killing by the bacterium. A targeting assay testing the colocalization of L. pneumophila-containing vacuole with an endoplasmic reticulum/pre-Golgi intermediate compartment marker protein, GFP-HDEL, was used to analyse these defects. In parental D. discoideum, the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min after introduction of bacteria to the amoebae. By 6 h after infection it was clear that the rtoA mutant acquired and retained the GFP-HDEL less efficiently than the parental strain, and that the mutant was defective for promoting the physical expansion of the membranous compartment surrounding the bacteria. Depressed intracellular growth of L. pneumophila in a D. discoideum rtoA(-) mutant therefore appeared to result from a lowered efficiency of vesicle trafficking events that are essential for the modification and expansion of the L. pneumophila-containing compartment.     

cAMP signaling in <Emphasis Type="Italic"> Aspergillus fumigatus </Emphasis>is involved in the regulation of the virulence gene <Emphasis Type="Italic"> pksP </Emphasis>and in defense against killing by macrophages

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease and resulting in high mortality. In order to determine the importance of the cAMP signaling pathway for virulence, three genes encoding putative elements of the pathway have been cloned and characterized: the adenylate cyclase gene acyA, and gpaA and gpaB, both of which encode alpha subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the deltaacyA mutant producing very few conidia. The growth rate of the deltaacyA mutant was also reduced, in contrast to that of the deltagpaB mutant. Addition of 10 mM dibutyryl-cAMP to the culture medium completely restored the wild-type phenotype in both mutant strains. To study the influence of GPAB on the expression of the gene pksP, which encodes a virulence factor that is involved in pathogenicity, a pksPp-lacZ gene fusion was generated and integrated as a single copy at the pyrG gene locus of both the parental strain and the deltagpaB mutant strain. The deltagpaB mutant showed reduced expression of the pksPp-lacZ reporter gene relative to that in the parental strain. In mycelia of both the parental strain and the deltagpaB mutant pksPp-lacZ expression was increased when isobutyl-methyl-xanthine, an inhibitor of intracellular phosphodiesterases, was added to the medium. The survival rate of conidia after ingestion by human monocyte-derived macrophages was also determined. The killing rate for conidia from deltaacyA and deltagpaB strains was significantly higher than that for wild-type conidia. Taken together, these findings suggest that cAMP triggers a system that protects A. fumigatus from the effects of immune effector cells of the host.     

Physico- and immunochemical properties of staphylococcal protein A extracellularly produced by a set of mutants from Staphylococcus aureus Cowan I.

Mutant strains of Staphylococcus aureus have been isloated using a simple cosedimentation technique. While the parental strain contains predominantly a cell-bound protein A, the mutant strains exclusively produce extracellular protein A. The mutant forms of protein A all have lower molecular weights than that of protein A from the parental strain. They showed the same antigenicity as the parental protein A and gave similar reactivity with immunoglobulin to the parental one except for one mutant. A conspicuous spur was observed between the parental protein A and that produced by the mutant against normal dog serum in the micro-Ouchterlony immunodiffusion test.     

Analysis of Different Types of Competitive Capacity in the Alfalfa Rhizobia (Sinorhizobium meliloti) Tn5 Mutants

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti.The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. melilotiRmM4gustester strain carrying the gene of -glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in plantaandex plantaecological niches.     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.     

Enhanced acid tolerance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> by adaptive evolution and compared stress response during acid stress

This study aimed to improve the acid tolerance of Lactobacillus casei Zhang and compare the stress response of the parental strain and the acid-resistant mutant during acidic conditions. Adaptive evolution was conducted for 70 days to generate acid-tolerant L. casei. The evolved mutant lb-2 exhibited more than a 60% increase in biomass as well as a 13.6 and 65.6% increase in concentrations of lactate and acetate, respectively, when cultured at pH 4.3 for 64 h. Lactic acid tolerances of the parental strain and the evolved mutant were determined. As a result, the evolved mutant showed a 318-fold higher survival rate than that of the parental strain. Physiological analysis showed that the evolved mutant exhibited higher intracellular pH (pH_i), NH₄ ⁺ concentration and lower inner membrane permeability than that of the parental strain during acid stress. Moreover, higher amounts of intracellular arginine and aspartate were also detected in lb-2 under acid stress. Validation of the relationship between the acid tolerance and the intracellular arginine and aspartate accumulation was conducted by experiments that showed the survival of L. casei at pH 3.3 was improved 1.36-, 2.10-, or 3.42-fold by the addition of 50 mM aspartate, arginine or both of them, respectively. Taken together, results presented here not only supply an effective way to select acid-resistant strains for the food industry, but also contribute to reveal the mechanisms of acid tolerance and provide new strategies to enhance the industrial utility and health-promoting properties of this species.     

A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Construction and properties of an intracellular serine protease mutant of Bacillus subtilis.

An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene. Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal. The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain. These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.