Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning in Micromonospora sp. Strain 40027

Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage ΦC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.     

Isolation and characterization of Micromonospora phage PhiHAU8 and development into a phasmid

PhiHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The PhiHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. PhiHAU8 was most stable at 4 degrees C in Difco nutrient broth within a pH range of 6 to 12. PhiHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca(2+) and 30 mM Mg(2+). A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a lambda cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that PhiHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.     

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)₈-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.     

Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn²⁺ or Mn²⁺ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M_r of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in B. subtilis cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.     

Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).     

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8⁺ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8⁺ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8⁺ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8⁺ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8⁺ T-cells segregated into IFNγ⁺ perforin (Pfn)^neg or IFNγ^negPfn⁺ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8⁺Pfn⁺ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8 ^−/− recipients showed that the CD8⁺ cells from infected ifnγ^−/− pfn ^+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8⁺ cells from ifnγ ^+/+ pfn ^−/− donors. Moreover, the reconstitution of naïve cd8 ^−/− mice with CD8⁺ cells from naïve ifnγ ^+/+ pfn ^−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ⁺ cells accumulation, whereas reconstitution with CD8⁺ cells from naïve ifnγ ^−/− pfn ^+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn⁺ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8⁺Pfn⁺ and CD8⁺IFNγ⁺ cells during CCC. CD8⁺IFNγ⁺ cells may exert a beneficial role, whereas CD8⁺Pfn⁺ may play a detrimental role in T. cruzi-elicited heart injury.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

Background

Antigen-specific IFN-γ producing CD4⁺ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-γ production without affecting protective IFN-γ is a challenge in tuberculosis research.

Methods and Findings

Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4⁺ T cell-mediated IFN-γ response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-γ response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8⁺ T cells which suppressed IFN-γ-secreting CD4⁺ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-γ responses by CD4⁺ T cells in protein-boosted mice without affecting the low protective IFN-γ-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8⁺ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-γ inhibition did not require soluble IL-10, TGF-β, XCL-1 and MIP-1β. In vivo Ag85B stimulation induced 4-1BB expression on CD8⁺ T cells and in vivo 4-1BB ligation reduced the activation, IFN-γ production and expansion of Ag85B-specific CD4⁺ T cells of DNA-primed and protein-boosted mice.

Conclusion/Significance

Antigen-specific suppressor CD8⁺ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-γ-secreting CD4⁺ T cells. The selective expression of 4-1BB only on CD8⁺ T cells in mice developing a massive, non-protective IFN-γ response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.     

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The K_m values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl₂. Conversion activity from β-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol. 148: 131-142, 1981) that γ-tsr-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of “heterogeneous” (HET) fragments in restriction enzyme digests of γ-tsr-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the γ phage genome as revealed in heteroduplexes of DNA from γ-tsr-1 and β corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of γ-tsr-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (attP), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced γ-tsr-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of γ-tsr-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the tox (diphtheria toxin) gene of γ-tsr-1 and γ-tsr-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that γ wild-type phage, which is mutant for tox, was originally tox⁺, but that transposition of bacterial DNA into the gene inactivated it.     

M-DNA is stabilised in G*C tracts or by incorporation of 5-fluorouracil

M-DNA is a complex between the divalent metal ions Zn²⁺, Ni²⁺ and Co²⁺ and duplex DNA which forms at a pH of ~8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl₂. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from T_m measurements. It is evident that the lower the pK_a of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion.     

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α₂ structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The K_m values for coniferyl aldehyde and NAD⁺ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH₂-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD⁺-dependent aldehyde dehydrogenases from different sources.     

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe²⁺, Ca²⁺, and Mg²⁺ greatly increased the xylanase activity, whereas Mn²⁺ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Autocrine Production of β-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4⁺ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4⁺ T cells, which are less frequently infected than HIV-specific CD4⁺ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4⁺ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4⁺ T cells rapidly up-regulated production of MIP-1α and MIP-1β mRNA, resulting in a rapid increase in production of MIP-1α and MIP-1β after cognate antigen stimulation. Production of β-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4⁺ T cells. To test whether production of β-chemokines by CD4⁺ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4⁺ T cells. We found that CMV-specific CD4⁺ T cells which produced MIP-1β contained 10 times less Gag DNA than did those which failed to produce MIP-1β. These data suggest that CD4⁺ T cells which produce MIP-1α and MIP-1β bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.     

Analysis of Mycobacterium tuberculosis-Specific CD8 T-Cells in Patients with Active Tuberculosis and in Individuals with Latent Infection

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-γ and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA⁺CCR7⁻). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2⁺/IFN-γ⁺ and IL-2⁻/IFN-γ⁺ T-cell populations; interestingly, only the IL-2⁺/IFN-γ⁺ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).     

Mobilization of Genomic Islands of Staphylococcus aureus by Temperate Bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Mechanisms of Action of Carvacrol on the Food-Borne Pathogen Bacillus cereus

Carvacrol, a naturally occurring compound mainly present in the essential oil fraction of oregano and thyme, was studied for its effect on bioenergetic parameters of vegetative cells of the food-borne pathogen Bacillus cereus. Incubation for 30 min in the presence of 1 to 3 mM carvacrol reduced the viable cell numbers exponentially. Carvacrol (2 mM) significantly depleted the intracellular ATP pool to values close to 0 within 7 min. No proportional increase of the extracellular ATP pool was observed. Depletion of the internal ATP pool was associated with a change of the membrane potential (Δψ). At concentrations of 0.01 mM carvacrol and above, a significant reduction of Δψ was observed, leading to full dissipation of Δψ at concentrations of 0.15 mM and higher. Finally, an increase of the permeability of the cytoplasmic membrane for protons and potassium ions was observed (at 0.25 and 1 mM carvacrol, respectively). From this study, it could be concluded that carvacrol interacts with the membranes of B. cereus by changing its permeability for cations like H⁺ and K⁺. The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.