Densities and inhibitory phenotypes among indigenous Streptomyces spp. vary across native and agricultural habitats.

Streptomyces spp. perform vital roles in natural and agricultural soil ecosystems including in decomposition and nutrient cycling, promotion of plant growth and fitness, and plant disease suppression. Streptomyces densities can vary across the landscape, and inhibitory phenotypes are often a result of selection mediated by microbial competitive interactions in soil communities. Diverse environmental factors, including those specific to habitat, are likely to determine microbial densities in the soil and the outcomes of microbial species interactions. Here, we characterized indigenous Streptomyces densities and inhibitory phenotypes from soil samples (n = 82) collected in 6 distinct habitats across the Cedar Creek Ecosystem Science Reserve (CCESR; agricultural, prairie, savanna, wetland, wet-woodland, and forest). Significant variation in Streptomyces density and the frequency of antagonistic Streptomyces were observed among habitats. There was also significant variation in soil chemical properties among habitats, including percent carbon, percent nitrogen, available phosphorus, extractable potassium, and pH. Density and frequency of antagonists were significantly correlated with one or more environmental parameters across all habitats, though relationships with some parameters differed among habitats. In addition, we found that habitat rather than spatial proximity was a better predictor of variation in Streptomyces density and inhibitory phenotypes. Moreover, habitats least conducive for Streptomyces growth and proliferation, as determined by population density, had increased frequencies of inhibitory phenotypes. Identifying environmental parameters that structure variation in density and frequency of antagonistic Streptomyces can provide insight for determining factors that mediate selection for inhibitory phenotypes across the landscape.

Pathogen variation and urea influence selection and success of Streptomyces mixtures in biological control.

Success in biological control of plant diseases remains inconsistent in the field. A collection of well-characterized Streptomyces antagonists (n = 19 isolates) was tested for their capacities to inhibit pathogenic Streptomyces scabies (n = 15 isolates). There was significant variation among antagonists in ability to inhibit pathogen isolates and among pathogens in their susceptibility to inhibition. Only one antagonist could inhibit all pathogens, and antagonist-pathogen interactions were highly specific, highlighting the limitations of single-strain inoculum in biological control. However, the collection of pathogens could be inhibited by several combinations of antagonists, suggesting the potential for successful antagonist mixtures. Urea generally increased effectiveness of antagonists at inhibiting pathogens in vitro (increased mean inhibition zones) but its specific effects varied among antagonist-pathogen combinations. In greenhouse trials, urea enhanced the effectiveness of antagonist mixtures relative to individual antagonists in controlling potato scab. Although antagonist mixtures were frequently antagonistic in the absence of urea, all n= 2 and n = 3 antagonist-isolate combinations were synergistic in the presence of urea. This work provides insights into the efficacy of single- versus multiple-strain inocula in biological control and on the potential for nutrients to influence mixture success.

Products of US22 genes M140 and M141 confer efficient replication of murine cytomegalovirus in macrophages and spleen.

Efficient replication of murine cytomegalovirus (MCMV) in macrophages is a prerequisite for optimal growth and spread of the virus in its natural host. Simultaneous deletion of US22 gene family members M139, M140, and M141 results in impaired replication of MCMV in macrophages and mice. In this study, we characterized the proteins derived from these three genes and examined the impact of individual gene deletions on viral pathogenesis. The M139, M140, and M141 gene products were identified as early proteins that localize to both the nucleus and cytoplasm in infected cells. Gene M139 encodes two proteins, of 72 and 61 kDa, while M140 and M141 each encode a single protein of 56 (pM140) and 52 (pM141) kDa, respectively. No role for the M139 proteins in MCMV replication in macrophages or mice was determined in these studies. In contrast, deletion of either M140 or M141 resulted in impaired MCMV replication in macrophages and spleen tissue. Replication of the M140 deletion mutant was significantly more impaired than that of the virus lacking M141. Further analyses revealed that the absence of the pM140 adversely affected pM141 levels by rendering the latter protein unstable. Since the replication defect due to deletion of M140 was more profound than could be explained by the reduced half-life of pM141, pM140 must exert an additional, independent function in mediating efficient replication of MCMV in macrophages and spleen tissue. These data indicate that the US22 genes M140 and M141 function both cooperatively and independently to regulate MCMV replication in a cell type-specific manner and, thus, to influence viral pathogenesis.

Transcriptional analysis of the murine cytomegalovirus HindIII-I region: identification of a novel immediate-early gene region.

Cytomegaloviruses likely encode numerous gene products involved in regulating virus-host cell interactions and pathogenesis. We previously identified a region of murine cytomegalovirus (MCMV) within HindIII-J and -I that regulates pathogenesis of the virus [open reading frames (ORFs) M139-M141] or is likely required for MCMV replication (ORFs m142 and m143). As a prerequisite for further studies on the structure and function of this gene region, we mapped the transcripts encoded within MCMV HindIII-I. Probes for ORFs M140 and M141 hybridized to 5.4- and 7.0-kb RNA, respectively, which were transcribed with early kinetics and were 3' coterminal with HindIII-J ORF M139. Probes representing ORFs m142, m143, or m144 hybridized to 3' coterminal transcripts of 1.8, 3.8, and 5.1 kb, respectively. ORFs m142 and m143 were transcribed with immediate-early kinetics but were most abundantly expressed at early times. Probes for the rightmost end of HindIII-I hybridized to a 5. 1-kb early/late RNA corresponding to m144 and to a 1.8-kb early RNA transcribed from m145. All of the major transcripts were polyadenylated and therefore are likely coding. Additional minor transcripts of intermediate sizes were also detected. ORFs M139-m143 showed homology to the betaherpesvirus-specific HCMV US22 gene family. Because deletion of these viral genes results in attenuated or helper-dependent phenotypes, this conserved region of US22 family genes may have a role in virus replication as well as in the pathogenesis of betaherpesviruses in their natural hosts.

Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis.

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains.

Reduction of major histocompatibility complex class I cell surface expression occurs in adenovirus-, herpes simplex virus-, human cytomegalovirus (HCMV)-, and murine cytomegalovirus-infected cell systems. Recently, it was demonstrated that the down-regulation mediated by HCMV infection is posttranslational, as a result of increased turnover of class I heavy chains in the endoplasmic reticulum (M. F. C. Beersma, M. J. E. Bijlmakers, and H. L. Ploegh, J. Immunol. 151:4455-4464, 1993; Y. Yamashita, K. Shimokata, S. Saga, S. Mizuno, T. Tsurumi, and Y. Nishiyama, J. Virol. 68:7933-7943, 1994. To identify HCMV genes involved in class I regulation, we screened our bank of HCMV deletion mutants for this phenotype. A mutant with a 9-kb deletion in the S component of the HCMV genome (including open reading frames IRS1 to US9 and US11) failed to down-regulate class I heavy chains. By examining the effects of smaller deletions within this portion of the HCMV genome, a 7-kb region containing at least nine open reading frames was shown to contain the genes required for reduction in heavy-chain expression. Furthermore, it was determined that at least two independent loci within the 7-kb region were able to cause class I heavy-chain down-regulation. One of these, US11, encodes a 32-kDa glycoprotein which causes down-regulation of class I heavy chains in the absence of other viral gene products. Hence, a specific function associated with a phenotype of the HCMV replicative cycle has been mapped to a dispensable gene region. These loci may be important for evasion of the host's immune response and viral persistence.

The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses.

Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH, and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory tract disease associated with influenza A/equine/Saskatoon/1/91 (H3N8), infections. Concentrations of antibodies binding to influenza A/equine/Kentucky/1/81 (H3N8), A/equine/Miami/1/63 (H3N8), and A/equine/Prague/1/56 (H7N7) were determined. Results of the VN assay were compared with results from the SRH and HI assays for acute antibody levels, changes in antibody concentrations between acute and convalescent sampling, and the occurrence of seroconversion. The correlation between assays for pre-exposure antibody levels ranged from 88% to 96%. The correlation between assays for change in antibody concentration ranged from 83% to 90% for the H3N8 viruses. This study shows that antibody concentrations specific for equine influenza virus, measured using SRH and HI assays, are highly correlated with concentrations detected using a VN assay.

Redox pathways in electron-transfer proteins: correlations between reactivities, solvent exposure, and unpaired-spin-density distributions.

The relative reactivities toward reduction by free flavin semiquinones of cytochromes (c-type cytochromes, cytochrome b5, c'-type cytochromes) iron-sulfur proteins (high-redox-potential ferredoxins, rubredoxins, low-redox-potential ferredoxins), and blue copper proteins (plastocyanin, azurins) are shown to correlate with calculations of the solvent exposure of the various prosthetic groups. In the case of the c-type cytochromes, one of the major centers of exposure is the sulfur atom of the thioether bridge that covalently links heme ring C to the protein. Charge-iterative extended Hückel calculations on a heme c model indicate that both porphyrin pi and Fe(III)d pi orbitals can delocalize onto the bridging sulfur atom. Unpaired spin densities are comparable to those obtained for individual aromatic porphyrin ring carbon atoms. Thus, the exposed sulfur of ring C may act to facilitate electron transfer.

Molecular complex of lumiflavin and 2-aminobenzoic acid: crystal structure, crystal spectra, and solution properties.

The molecular complex lumiflavin-2-aminobenzoic acid monohydrate (C13H12N4O2.C7H7NO2.H2O) crystallizes from from aqueous solution as red triclinic prisms. The space group is P1 with cell dimensions a = 9.660 A, b = 14.866 A, c = 7.045 A, alpha = 95.44 degrees , beta = 95.86 degrees, and gamma = 105.66 degrees . The crystal structure was solved by direct methods and refined by block-diagonal least-squares procedures to an R value of 0.050 on the basis of 1338 observed reflections. The structure is composed of stacks of alternating lumiflavin adn un-ionized (neutral) 2-aminobenzoic acid molecules. Two different modes of stacking interaction are observed. In one, 2-aminobenzoic acid overlaps all three of the isoalloxazine rings, at a mean distance of 3.36 A; in the other, 2-aminobenzoic acid interacts distance of 3.36 A; in the other, 2-aminobenzoic acid interacts with the pyrazine and dimethylbenzene moieties, at a distance of 3.42 A. Perpendicular to the stacking direction, the molecules form a continuous sheet. Each flavin is hydrogen bonded via O(2) and NH(3) to two symmetrically related aminobenzoates; the water of crystallization forms three hydrogen bonds, bridging two flavins, via O(4) and N(5), and one aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid molecules. Measurements of the polarized optical absorption spectra of crystals show that the transition moment direction for the long wavelength absorbance (beyond 530 nm) contains an out-of-plane component which can only arise from a charge-transfer interaction. Since the amino N does not make exceptionally close interactions with isoalloxazine atoms in either stacking mode (minimum interatomic distance 3.52 A), the charge transfer is presumed to involve pi orbitals of the 2-aminobenzoic acid donor.

pi cation radicals of ferrous and free base isobacteriochlorins: Models for siroheme and sirohydrochlorin.

Theoretical and experimental optical, redox, and paramagnetic results are presented for models of siroheme, the iron isobacteriochlorin prosthetic group of nitrite and sulfite reductases, and of sirohydrochlorin, the metal-free siroheme that is an intermediate in the biosynthetic pathway to vitamin B(12). The facile oxidation of many isobacteriochlorins, which distinguishes them from porphyrins and chlorins, suggests that the siroheme macrocycle itself may undergo oxidation in the multi-electron enzymatic cycles that reduce nitrite to ammonia and sulfite to hydrogen sulfide. Extended Hückel MO calculations (i) help rationalize the redox properties of isobacteriochlorins compared with those of porphyrins and chlorins; (ii) indicate that Fe(II) pyridine carbonyl[(py) (CO)] complexes of isobacteriochlorins, unlike those of porphyrins and chlorins, should undergo oxidation from the macrocycle rather than the metal to yield pi cation radicals; (iii) suggest that, in hexacoordinated Fe(II) isobacteriochlorin complexes, the site of oxidation-i.e., the metal or the macrocycle-will depend on the ligand field induced by the axial ligands; and (iv) predict similar unpaired spin density profiles for metal-free and (py) (CO)Fe(II) isobacteriochlorin radicals. Experimental data for three isomeric free-base and (py) (CO)Fe(II) complexes of dimethyloctaethylisobacteriochlorins support the theoretical calculations and establish the existence of Fe(II) isobacteriochlorin pi cations in vitro.