Biochemical Characterization of Haloalkane Dehalogenases DrbA and DmbC,Representatives of a Novel Subfamily

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.Haloalkane dehalogenases (EC 3.8.1.5.) acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (4, 6, 22), construction of biosensors (2), decontamination of warfare agents (17), and synthesis of optically pure compounds (19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (3). In contrast to subfamilies HLD-I and HLD-II, the subfamily HLD-III is currently lacking experimentally characterized proteins. We have therefore focused on the isolation and study of two selected representatives of the HLD-III subfamily, DrbA and DmbC.The drbA gene was amplified by PCR using the cosmid pircos.a3g10 originating from marine bacterium Rhodopirellula baltica SH1, and the dmbC gene was amplified from DNA originating from obligatory pathogen Mycobacterium bovis 5033/66. Six-histidine tails were added to the C termini of DrbA and DmbC in a cloning step, enabling single-step purification using Ni-nitrilotriacetic acid resin. Haloalkane dehalogenase DrbA was expressed under the T7 promoter and purified, with a resulting yield of 0.1 mg of protein per gram of cell mass. Haloalkane dehalogenase DmbC was obtained by expression in Mycobacterium smegmatis, with a yield of 0.07 mg of purified protein per gram of cell mass.The correct folding and secondary structures of the newly prepared enzymes were verified by circular dichroism (CD) spectroscopy. Far-UV CD spectra were recorded for DrbA and DmbC enzymes and other, related haloalkane dehalogenases. All enzymes tested exhibited CD spectra with two negative features at 208 and 222 nm and one positive peak at 195 nm, which are characteristic of α-helical content (Fig. ​(Fig.1).1). This suggested that both new enzymes, DrbA and DmbC, were folded correctly. However, DmbC exhibited more intense negative maxima which differed from other haloalkane dehalogenases in the θ₂₂₂/θ₂₀₈ ratio. This finding indicated a slight variation in the arrangement of secondary structure elements of the DmbC enzyme. Thermally induced denaturations of DrbA and DmbC were tested in parallel. Both enzymes showed changes in ellipticity during increasing temperature. The melting temperatures calculated from these curves were 45.8 ± 0.4°C for DmbC and 39.4 ± 0.1°C for DrbA. The thermostability results obtained for DrbA and DmbC were in good agreement with the range of melting temperatures determined for other, related haloalkane dehalogenases.Open in a separate windowFIG. 1.Far-UV CD spectra of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Protein concentration used for far-UV CD spectrum measurement was 0.2 mg/ml.The sizes of the purified proteins were estimated by electrophoresis under native conditions conducted using a 10% polyacrylamide gel (Fig. ​(Fig.2).2). More precise determination of the sizes of DrbA and DmbC was achieved by gel filtration chromatography performed on Sephacryl S-500 HR (GE Healthcare, Uppsala, Sweden), calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (Fig. ​(Fig.3A).3A). Both DrbA and DmbC were eluted from the column in the fraction prior to blue dextran, indicating that both enzymes form oligomeric complexes of a size larger than 2,000 kDa (Fig. 3B and C). The haloalkane dehalogenases which have been biochemically characterized so far form monomers, except for DbjA isolated from Bradyrhizobium japonicum USDA110 (21), which shows monomeric, dimeric, and tetrameric forms according to the pH of the buffer (R. Chaloupkova, submitted for publication).Open in a separate windowFIG. 2.Native protein electrophoresis of DrbA and DmbC. Lane 1, carbonic anhydrase (29 kDa); lane 2, ovalbumin (43 kDa); lane 3, bovine albumin (67 kDa); lane 4, conalbumin (75 kDa); lane 5, catalase (240 kDa); lane 6, ferritin (440 kDa); lane 7, DrbA; lane 8, DmbC.Open in a separate windowFIG. 3.Gel filtration chromatogram of DrbA and DmbC. (A) The following calibration kit samples (0.5 ml of a concentration of 2 mg/ml protein loaded) were analyzed using 50 mM Tris-HCl with 150 mM NaCl, pH 7.5, as elution buffer: blue dextran (line 1, 9.6-ml fraction), thyroglobulin (line 2, 15.95-ml fraction), ferritin (line 3, 16.78-ml fraction), ovalbumin (line 4, 18.55-ml fraction), and RNase A (line 5, 20.08-ml fraction). (B and C) Haloalkane dehalogenase DrbA eluted in the 9.03-ml fraction (B), and haloalkane dehalogenase DmbC in the 9.31-ml fraction (C).The substrate specificities of DrbA and DmbC were investigated with a set of 30 selected chlorinated, brominated, and iodinated hydrocarbons. Standardized specific activities related to 1-chlorobutane (summarized in Table ​Table1)1) were compared with the activity profiles of other haloalkane dehalogenases (Fig. ​(Fig.4).4). DrbA and DmbC displayed similar activity patterns, with catalytic activities approximately two orders of magnitude lower than those of other known haloalkane dehalogenases (1, 5, 8-11, 13-16, 18, 20, 23). HLD-III subfamily enzymes showed a restricted specificity range and a preference for iodinated short-chain hydrocarbons. Both phenomena may be related to the composition of the catalytic pentad Asp-His-Asp+Asn-Trp, which is unique to the members of the HLD-III subfamily (3). The preference for substrates carrying an iodine substituent can be related to a pair of halide-binding residues and their spatial arrangement with the catalytic triad. These residues make up the catalytic pentad, playing a critical role in substrate binding, formation of the transition states, and the reaction intermediates of the dehalogenation reaction (12).Open in a separate windowFIG. 4.Substrate specificity profiles of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Activities were determined using a consistent set of 30 halogenated substrates (see Table ​Table1).1). Data were standardized by dividing each value by the sum of all activities determined for individual enzymes in order to mask the differences in absolute activities. Specific activities (in μmol·s⁻¹·mg⁻¹) with 1-chlorobutane are 0.0003 (DrbA), 0.0001 (DmbC), 0.0003 (DatA), 0.0133 (DbjA), 0.0010 (DbeA), 0.0128 (DhaA), 0.0231 (LinB), 0.0171 (DmbA), and 0.0117 (DhlA).

TABLE 1.

Specific activities of haloalkane dehalogenases DrbA and DmbC toward a set of 30 halogenated hydrocarbons^a

Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution

Hepatitis C virus (HCV)-specific CD8⁺ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127⁺ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4⁺ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8⁺ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables ​(Tables11 and ​and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP^Sc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.PrP^Sc, a disease-specific marker for prion diseases and the likely infectious agent, is widely distributed within the central nervous system (CNS) and lymphoreticular tissues (LRS) in ovine scrapie, human variant Creutzfeldt-Jakob disease (vCJD), and cervine chronic wasting disease (CWD) during both clinical and preclinical stages (4, 11, 25). Furthermore, while the LRS distribution of PrP^Sc is much more restricted in bovine spongiform encephalopathy (BSE), sheep experimentally infected with BSE display a PrP^Sc tissue distribution more akin to that of ovine scrapie (11).For rodent-adapted scrapie and cervine CWD, the disease agent is detected in excreta when animals are in the clinical stages of disease, a process likely to contribute to environmental reservoirs of infectivity and lateral disease transmission (5, 13, 21). Within an experimental rodent model, it has also been demonstrated that the shedding of PrP^Sc and concomitant infectivity in feces occurs during preclinical scrapie (21).Evidence now also demonstrates that milk provides a vehicle for the transmission for prion diseases. Scrapie-free lambs fed milk from clinical scrapie-affected ewes propagate PrP^Sc within their LRS (8). Additionally, a recent study using a transgenic mouse bioassay demonstrated the secretion of infectivity in milk from preclinical animals where scrapie infectivity was found in milk months before the onset of clinical signs in animals with an ARQ/VRQ PrP genotype (10). The presence of scrapie infectivity within milk was irrespective of mammary gland pathology or PrP^Sc accumulation, and these animals were estimated to have considerable accumulation of immunohistochemically (IHC) detectable PrP^Sc within the LRS at the time of sampling.Here, we applied serial protein misfolding cyclic amplification (sPMCA) to the in vitro detection of PrP^Sc within sheep milk (Fig. ​(Fig.1)1) (Table ​(Table11).Open in a separate windowFIG. 1.sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrP^Sc negative until the stated round and thereafter was positive.

TABLE 1.

Timeline of exposure of animals to a scrapie-positive farm environment, sample collection, and scrapie status

Cross-Subtype Neutralization Sensitivity despite Monoclonal Antibody Resistance among Early Subtype A,C, and D Envelope Variants of Human Immunodeficiency Virus Type 1

The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.Most effective viral vaccines are thought to provide protection primarily by stimulating neutralizing antibodies (NAbs) to clear cell-free virus (25, 27). Because protection by NAbs requires recognition of common viral epitopes, the extreme genetic diversity of human immunodeficiency virus type 1 (HIV-1) presents a particular challenge to NAb-based vaccine approaches. Therefore, a critical starting point for studies of immune-mediated protection against HIV-1 is a collection of newly transmitted HIV-1 variants, particularly from areas of endemicity, such as sub-Saharan Africa, in order to determine whether vaccines are appropriately targeted to common epitopes from these relevant transmitted strains.During HIV-1 transmission, a bottleneck allows only one or a few variants to be transmitted to a newly infected individual (6, 9, 16, 29, 34, 37, 39), and the sensitivity of these early transmitted strains to antibody-mediated neutralization is therefore of particular interest. Newly transmitted HIV-1 variants have demonstrated significant heterogeneity in their neutralization phenotypes both within and between subtypes (2, 3, 6-8, 11, 13-15, 22, 30, 32, 36). Panels of sexually transmitted HIV-1 envelope variants (based on the envelope gene, env) have been characterized, including subtype B variants from North America, Trinidad, and Europe, subtype C variants from South Africa and Zambia, and subtype A variants from Kenya collected between 1994 and 1996 (2, 14, 15). Here, we characterize an additional 31 envelope variants from 14 subjects with sexually transmitted HIV-1 who were infected in Kenya, where subtypes A, C, and D circulate, between 1993 and 2005 (24, 31).The env genes were cloned from samples drawn 14 to 391 (median, 65) days postinfection from individuals enrolled in a prospective cohort of high-risk women in Mombasa, Kenya (19-21). Demographic characteristics of the subjects are summarized in Table ​Table1;1; the timing of first infection was determined by both HIV-1 serology and HIV RNA testing as described previously (12). All of the subjects were presumably infected by male-to-female transmission and displayed a range of plasma viral loads at the time of env gene cloning (Table ​(Table1).1). For most individuals, full-length env genes were cloned from uncultured peripheral blood mononuclear cell (PBMC) DNA, though for two individuals, clones were obtained from DNA following short-term coculture with donor PBMCs (Table ​(Table1).1). env genes were cloned by single-copy nested PCR with primers and PCR conditions as described previously (4, 17). We tested env genes for their ability to mediate infection by transfecting env plasmid DNA into 293T cells along with an env-deficient HIV-1 subtype A proviral plasmid, Q23Δenv, to make pseudoviral particles (17). More than 80 env clones were obtained from 16 subjects; less than one-half were functional on the basis of the infectivity of pseudoviral particles in a single-round infection of TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health), as observed previously for env genes cloned from proviral sequences (17); a lower fraction of functional env genes have been reported from plasma (18). We focused on the proviral sequences here because they presumably best represent the sequence closest to that of the transmitted strains. The 31 functional env variants are described in Table ​Table11.

TABLE 1.

Demographic characteristics, diversities, gp120 variable-region lengths, numbers of PNGS, and accession numbers of cloned env variants

Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens. IV. Genetic Constraints Guide Evolutionary Trajectories in a Parallel Adaptive Radiation

The capacity for phenotypic evolution is dependent upon complex webs of functional interactions that connect genotype and phenotype. Wrinkly spreader (WS) genotypes arise repeatedly during the course of a model Pseudomonas adaptive radiation. Previous work showed that the evolution of WS variation was explained in part by spontaneous mutations in wspF, a component of the Wsp-signaling module, but also drew attention to the existence of unknown mutational causes. Here, we identify two new mutational pathways (Aws and Mws) that allow realization of the WS phenotype: in common with the Wsp module these pathways contain a di-guanylate cyclase-encoding gene subject to negative regulation. Together, mutations in the Wsp, Aws, and Mws regulatory modules account for the spectrum of WS phenotype-generating mutations found among a collection of 26 spontaneously arising WS genotypes obtained from independent adaptive radiations. Despite a large number of potential mutational pathways, the repeated discovery of mutations in a small number of loci (parallel evolution) prompted the construction of an ancestral genotype devoid of known (Wsp, Aws, and Mws) regulatory modules to see whether the types derived from this genotype could converge upon the WS phenotype via a novel route. Such types—with equivalent fitness effects—did emerge, although they took significantly longer to do so. Together our data provide an explanation for why WS evolution follows a limited number of mutational pathways and show how genetic architecture can bias the molecular variation presented to selection.UNDERSTANDING—and importantly, predicting—phenotypic evolution requires knowledge of the factors that affect the translation of mutation into phenotypic variation—the raw material of adaptive evolution. While much is known about mutation rate (e.g., Drake et al. 1998; Hudson et al. 2002), knowledge of the processes affecting the translation of DNA sequence variation into phenotypic variation is minimal.Advances in knowledge on at least two fronts suggest that progress in understanding the rules governing the generation of phenotypic variation is possible (Stern and Orgogozo 2009). The first stems from increased awareness of the genetic architecture underlying specific adaptive phenotypes and recognition of the fact that the capacity for evolutionary change is likely to be constrained by this architecture (Schlichting and Murren 2004; Hansen 2006). The second is the growing number of reports of parallel evolution (e.g., Pigeon et al. 1997; ffrench-Constant et al. 1998; Allender et al. 2003; Colosimo et al. 2004; Zhong et al. 2004; Boughman et al. 2005; Shindo et al. 2005; Kronforst et al. 2006; Woods et al. 2006; Zhang 2006; Bantinaki et al. 2007; McGregor et al. 2007; Ostrowski et al. 2008)—that is, the independent evolution of similar or identical features in two or more lineages—which suggests the possibility that evolution may follow a limited number of pathways (Schluter 1996). Indeed, giving substance to this idea are studies that show that mutations underlying parallel phenotypic evolution are nonrandomly distributed and typically clustered in homologous genes (Stern and Orgogozo 2008).While the nonrandom distribution of mutations during parallel genetic evolution may reflect constraints due to genetic architecture, some have argued that the primary cause is strong selection (e.g., Wichman et al. 1999; Woods et al. 2006). A means of disentangling the roles of population processes (selection) from genetic architecture is necessary for progress (Maynard Smith et al. 1985; Brakefield 2006); also necessary is insight into precisely how genetic architecture might bias the production of mutations presented to selection.Despite their relative simplicity, microbial populations offer opportunities to advance knowledge. The wrinkly spreader (WS) morphotype is one of many different niche specialist genotypes that emerge when experimental populations of Pseudomonas fluorescens are propagated in spatially structured microcosms (Rainey and Travisano 1998). Previous studies defined, via gene inactivation, the essential phenotypic and genetic traits that define a single WS genotype known as LSWS (Spiers et al. 2002, 2003) (Figure 1). LSWS differs from the ancestral SM genotype by a single nonsynonymous nucleotide change in wspF. Functionally (see Figure 2), WspF is a methyl esterase and negative regulator of the WspR di-guanylate cyclase (DGC) (Goymer et al. 2006) that is responsible for the biosynthesis of c-di-GMP (Malone et al. 2007), the allosteric activator of cellulose synthesis enzymes (Ross et al. 1987). The net effect of the wspF mutation is to promote physiological changes that lead to the formation of a microbial mat at the air–liquid interface of static broth microcosms (Rainey and Rainey 2003).Open in a separate windowFigure 1.—Outline of experimental strategy for elucidation of WS-generating mutations and their subsequent identity and distribution among a collection of independently evolved, spontaneously arising WS genotypes. The strategy involves, first, the genetic analysis of a specific WS genotype (e.g., LSWS) to identify the causal mutation, and second, a survey of DNA sequence variation at specific loci known to harbor causal mutations among a collection of spontaneously arising WS genotypes. For example, suppressor analysis of LSWS using a transposon to inactivate genes necessary for expression of the wrinkly morphology delivered a large number of candidate genes (top left) (Spiers et al. 2002). Genetic and functional analysis of these candidate genes (e.g., Goymer et al. 2006) led eventually to the identity of the spontaneous mutation (in wspF) responsible for the evolution of LSWS from the ancestral SM genotype (Bantinaki et al. 2007). Subsequent analysis of the wspF sequence among 26 independent WS genotypes (bottom) showed that 50% harbored spontaneous mutations (of different kinds; see Open in a separate windowFigure 2.—Network diagram of DGC-encoding pathways underpinning the evolution of the WS phenotype and their regulation. Overproduction of c-di-GMP results in overproduction of cellulose and other adhesive factors that determine the WS phenotype. The ancestral SBW25 genome contains 39 putative DGCs, each in principle capable of synthesizing the production of c-di-GMP, and yet WS genotypes arise most commonly as a consequence of mutations in just three DGC-containing pathways: Wsp, Aws, and Mws. In each instance, the causal mutations are most commonly in the negative regulatory component: wspF, awsX, and the phosphodiesterase domain of mwsR (see text).To determine whether spontaneous mutations in wspF are a common cause of the WS phenotype, the nucleotide sequence of this gene was obtained from a collection of 26 spontaneously arising WS genotypes (WS_A-Z) taken from 26 independent adaptive radiations, each founded by the same ancestral SM genotype (Figure 1): 13 contained mutations in wspF (Bantinaki et al. 2007). The existence of additional mutational pathways to WS provided the initial motivation for this study.

TABLE 1

Mutational causes of WS

Evolutionary Strata in a Small Mating-Type-Specific Region of the Smut Fungus Microbotryum violaceum

DNA sequence analysis and genetic mapping of loci from mating-type-specific chromosomes of the smut fungus Microbotryum violaceum demonstrated that the nonrecombining mating-type-specific region in this species comprises ∼25% (∼1 Mb) of the chromosome length. Divergence between homologous mating-type-linked genes in this region varies between 0 and 8.6%, resembling the evolutionary strata of vertebrate and plant sex chromosomes.EVOLUTION of mating types or sex-determining systems often involves the suppression of recombination around the primary sex-determining or mating-type-determining locus. In animals and plants, it is often an entire or almost entire chromosome (Y or W in male or female heterogametic species, respectively) that ceases to recombine with its homologous (X or Z) chromosome (Charlesworth and Charlesworth 2000; Charlesworth 2008). Self-incompatibility loci in plants are also thought to be located in regions of suppressed recombination (Charlesworth et al. 2005; Kamau and Charlesworth 2005; Kamau et al. 2007; Li et al. 2007; Yang et al. 2007). Regardless of the phylogenetic position of a species, such nonrecombining regions are known to follow similar evolutionary trajectories. The nonrecombining region on the sex-specific chromosome expands in several steps, forming evolutionary strata—regions of different X/Y (or Z/W) divergence (Lahn and Page 1999; Handley et al. 2004; Sandstedt and Tucker 2004; Nicolas et al. 2005)—and genes in the nonrecombining regions gradually accumulate deleterious mutations that eventually render them dysfunctional (Charlesworth and Charlesworth 2005; Charlesworth 2008).Fungal mating-type systems are very diverse, with the number of mating types varying from two to several hundred (Casselton 2002). Like sex chromosomes in several animals and plants, suppressed recombination has evolved in regions near fungal mating-type loci, including in Ustilago hordei (Lee et al. 1999), Cryptococcus neoformans (Lengeler et al. 2002), and Neurospora tetrasperma (Menkis et al. 2008). These species have two mating types, but no morphologically distinct sexes. The mating-type locus (the region of suppressed recombination) of C. neoformans is small (∼100 kb) compared with known sex chromosomes and contains only ∼20 genes that, unlike many sex chromosomes (Y or W chromosomes), show no obvious signs of genetic degeneration (Lengeler et al. 2002; Fraser et al. 2004). Judging from the divergence between the homologous genes on the two mating-type-specific chromosomes, C. neoformans started to evolve sex chromosomes a long time ago because silent divergence between the two mating types in the most ancient region exceeds 100% (Fraser et al. 2004). Genes in the younger mating-type-specific region are much less diverged between the two sex chromosomes, suggesting that the evolution of the sex locus in C. neoformans might have proceeded through several steps. The nonrecombining region around the mating-type locus of N. tetrasperma is much larger than in C. neoformans (at least 6.6 Mb), and silent divergence between homologous genes on the mating-type-specific chromosomes ranges from zero to 9%, demonstrating that these mating-type-specific chromosomes evolved recently (Menkis et al. 2008).M. violaceum, which causes anther smut disease in Silene latifolia and other species in the family Caryophyllaceae, has two mating types, A1 and A2 (reviewed by Giraud et al. 2008), which are determined by the presence of mating-type-specific chromosomes (hereafter A1 and A2 chromosomes, or sex chromosomes) in the haploid stage of the life cycle (Hood 2002; Hood et al. 2004). The A1 and A2 chromosomes are distinguishable by size in pulsed-field electrophoresis, and it is possible to isolate individual chromosomes electrophoretically (Hood et al. 2004). Random fragments of A1 and A2 chromosomes have previously been isolated from mating-type-specific bands of pulsed-field separated chromosomes of M. violaceum (Hood et al. 2004). These fragments were assumed to be linked to mating type. The same method was used to isolate fragments of non-mating-type-specific chromosomes. On the basis of the analysis of their sequences, (Hood et al. 2004) proposed that mating-type-specific chromosomes in M. violaceum might be degenerate because they contained a lower proportion of protein-coding genes than other chromosomes. However, it was not determined whether the sequences isolated from the mating-type chromosomes originated from the mating-type-specific or from the recombining regions (Hood et al. 2004), and the relative sizes of these regions are not known for these M. violaceum chromosomes. We tested the mating-type specificity of 86 of these fragments and demonstrate that fewer than a quarter of these loci are located in the mating-type-specific region, suggesting that the nonrecombining region on the A1 and A2 chromosomes is quite small, while the rest of the chromosome probably recombines (like pseudoautosomal regions of sex chromosomes) and is therefore not expected to undergo genetic degeneration. Genetic mapping confirms the presence of two pseudoautosomal regions in the M. violaceum mating-type-specific chromosomes.As these chromosomes are mating type specific in the haploid stage of M. violaceum, mating-type-specific loci (or DNA fragments) can be identified by testing whether they are present exclusively in A1 or A2 haploid strains. We therefore prepared haploid A1 and A2 M. violaceum cultures from S. latifolia plants from two geographically remote locations (accessions Sl405 from Sweden and Sl127 from the French Pyrenees). Haploid sporidial cultures were isolated by a standard dilution method (Kaltz and Shykoff 1997; Oudemans and Alexander 1998). Mating types were determined by PCR amplification of each culture with primers designed for A1 and A2 pheromone receptor genes linked to A1 and A2 mating types (Yockteng et al. 2007). The primers were as follows: 5′-TGGCATCCCTCAATGTTTCC-3′ and 5′-CACCTTTTGATGAGAGGCCG-3′ for the A1 pheromone receptor (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF584742","term_id":"156254864","term_text":"EF584742"}}EF584742) and 5′-TGACGAGAGCATTCCTACCG-3′ and 5′-GAAGCGGAACTTGCCTTTCT-3′ for the A2 pheromone receptor (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF584741","term_id":"156254862","term_text":"EF584741"}}EF584741). Cultures with PCR product amplified only from an A1 or A2 pheromone receptor gene were selected for further use. The mating types of the cultures were verified by conjugating them in all combinations.The GenBank nucleotide database was searched using BLAST for sequences similar to those isolated by Hood et al. (2004). Sequences with similarity to transposable elements (TE) and other repeats were excluded. The resulting set of nonredundant sequences was used to design PCR primers for 98 fragments. Half of these were originally isolated from the A1 and half from the A2 chromosomes and are hereafter called A1-NNN or A2-NNN (where NNN is the locus number; supporting information, Table S1), which does not imply that these loci are A1 or A2 specific, but merely indicates that they were originally isolated from the A1 or A2 chromosomes. Amplification of these regions from new A1 and A2 M. violaceum cultures, independently isolated by ourselves, revealed that only 5 of the 49 loci isolated from the A1 chromosome are indeed A1 specific and only 6 of 49 isolated from the A2 chromosome are A2 specific. All other loci amplified from both A1 and A2 cultures. Figure 1 illustrates some of these results from the Swedish sample (Sl405).Open in a separate windowFigure 1.—Testing of mating-type specificity for loci isolated from A1 and A2 chromosomes. (a) PCR amplifications from haploid cultures from Sl405 using primers designed from six A1-originated loci. Loci in which a PCR product could be amplified only from A1 cultures (boxed) were classified as specific to mating type A1. (b) PCR tests of six A2-originated loci on the same set of haploids as in a. Loci in which a PCR product amplified only from A2 cultures (boxed) were classified as specific to mating type A2. Loci amplified from both A1 and A2 cultures are not mating type specific.The fragments that amplified from both A1 and A2 mating types may be in recombining regions, or they could be present in mating-type-specific regions on both A1 and A2 chromosomes. If they are in recombining regions, the A1- and A2-linked homologs should not be diverged from each other, but if they are in nonrecombining, mating-type-specific regions, the divergence of the A1- and A2-linked homologs should be roughly proportional to the time since recombination stopped in the region. We therefore sequenced and compared PCR fragments amplified from the two mating types of Sl405 or Sl127 cultures (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"FI855822","term_id":"258612459","term_text":"FI855822"}}FI855822–{"type":"entrez-nucleotide","attrs":{"text":"FI856001","term_id":"258612570","term_text":"FI856001"}}FI856001). Sequencing of PCR products showed that 12 (4 A1 and 8 A2) loci have more than one copy, and they were excluded from further analysis. Sequences of 61 loci were identical between the A1 and A2 strains, and four loci demonstrated low total divergence (0.24–0.61%) between the two mating types (otintseva and D. Filatov, unpublished results). Thus, these loci might be located in the recombining part of the mating-type-specific chromosomes. Ten of 75 loci that amplified in both mating types demonstrated multiple polymorphisms fixed between the mating types rather than between the locations. Given that the strains that we used in the analysis originated from two geographically distant locations, it is highly unlikely that multiple polymorphisms distinguishing the A1 and A2 sequences arose purely by chance; thus, these loci are probably located in the nonrecombining mating-type-specific region of the M. violaceum A1 and A2 chromosomes.

TABLE 1

Loci from mating-type-specific chromosomes of M. violaceum used for PCR analysis and genetic map construction

		With nonzero A1/A2 divergenceb
Loci	Mating type specific	<1%	>1%	With zero A1/A2 divergenceb	Total
A1a	5	2 (1)	3 (3)	35 (3)	45 (7)
A2a	6	2 (0)	7 (7)	26 (3)	41 (10)
Subtotal		4 (1)	10 (10)
Total	11	14 (11)		61 (6)	86 (17)

Open in a separate window^aA1, loci originated from the A1 sex chromosome; A2, loci originated from the A2 sex chromosome.^bThe number of loci used for genetic map construction is in parentheses.To confirm the mating-type-specific or pseudoautosomal locations of the loci with and without A1/A2 divergence, we conducted genetic mapping in a family of 99 individuals, 50 of which were of mating type A1 and 49 of mating type A2. The family was generated by a cross between A1 and A2 M. violaceum strains from S. latifolia accessions Sl405 (Sweden) and Sl127 (France), respectively. The choice of strains from geographically distant locations was motivated by the hope of maximizing the number of DNA sequence differences between them that can be used as molecular genetic markers in segregation analysis. We inoculated S. latifolia seedlings with sporidial cultures of both mating types. For inoculation, petri dishes with 12-day-old seedlings of S. latifolia were flooded with 2.5 ml of inoculum suspension. Inoculum suspension consisted of equal volumes of the A1 and A2 sporidial cultures that were mixed and conjugated overnight at 14° under rotation (Biere and Honders 1996; Van Putten et al. 2003). Seedlings were potted 3 days after inoculation. Two months later, teliospores were collected from the flowers of the infected plant and grown in petri dishes on 3.6% potato dextrose agar medium. Haploid sporidia formed after meiosis were isolated and grown as separate cultures for DNA extraction. The mating types of single sporidia cultures were identified as described above. The loci analyzed in the segregation analysis were sequenced in the two parental haploid strains and in 99 (50 A1 and 49 A2) haploid strains that were generated in the cross. Single nucleotide differences between the parental strains were used as molecular genetic markers for segregation analysis in the progeny. The genetic map was constructed using MAPMAKER/EXP v3.0 (Lincoln et al. 1992) and MapDisto v1.7 (http://mapdisto.free.fr/).The resulting genetic map is shown in Figure 2. As expected, no recombination was observed between the 10 loci with diverged A1- and A2-linked copies. In addition, one marker with no A1/A2 divergence, A2-397, was also completely linked to the loci with significant A1/A2 divergence. This locus either may be very tightly linked to the nonrecombining mating-type-specific region or may have been added to that region more recently than the loci that had already accumulated some divergence between the alleles in the two mating types. The mating-type-specific pheromone receptor locus (Devier et al. 2009) and 11 mating-type-specific loci are also located in this nonrecombining region (Figure 2). Interestingly, the cluster of nonrecombining markers is flanked on both sides with markers that recombine in meiosis, demonstrating that there are pseudoautosomal regions on both ends of the mating-type-specific chromosomes.Open in a separate windowFigure 2.—Genetic map of the mating-type-determining chromosome in M. violaceum. Genetic distance (in centimorgans) and the relative positions of the markers are shown to the left and the right of the chromosome, respectively. The position of the nonrecombining region corresponds to the cluster of linked markers shown on the right of the figure. Total A1/A2 divergence is shown in parentheses. Eleven mating-type-specific markers (for which sequences are available from only one mating type), located in the nonrecombining mating-type-specific region, are not shown.Our results demonstrate that although the loci reported by Hood et al. (2004) were isolated from the A1 and A2 chromosomes, most of these loci are not located in the nonrecombining mating-type-specific regions. In fact, the nonrecombining region might be relatively small: of 86 tested fragments, only 21 appeared to be either mating type specific or linked to the mating-type locus. Assuming that these loci represent a random set of DNA fragments isolated from the A1 and A2 chromosomes, it is possible to estimate the size of the nonrecombining region using the binomial distribution: the nonrecombining region is expected to be 24.4% (95% CI: 16.7–33.6%) of the chromosome length. As the sizes of the A1 and A2 chromosomes are ∼3.4 and 4.2 Mb long (Hood 2002; Hood et al. 2004), the nonrecombining region might be ∼1 Mb long.Interestingly, total A1/A2 divergence for the 11 loci with A1- and A2-linked copies mapped to the nonrecombining region varied from 0% to 8.6% (Figure 2). In addition, 11 loci amplified from only one mating type. These genes could represent degenerated genes, some of which degenerated in A1 strains, and some in A2 strains. Alternatively, they might be highly diverged genes, such that the PCR primers amplify only one allele, and not the other. Variation in divergence may be the result of the stepwise cessation of recombination between the A1 and A2 chromosomes in M. violaceum, resembling the evolutionary strata reported for human, chicken, and white campion sex chromosomes (Lahn and Page 1999; Handley et al. 2004; Bergero et al. 2007). However, only the differences between the most and the least diverged loci are statistically significant (Devier et al. 2009), the M. violaceum mating-type region has at least three strata: one oldest stratum, including the pheromone receptor locus; a younger stratum with ∼5–9% A1/A2 divergence; and the youngest stratum with 1–4% divergence between the two mating types. There may also be an additional very recently evolved stratum containing the locus named A2-397, which is also present in all A1 strains tested, with no fixed differences between the A1 and A2 strains (

Wide Variation in the Multiplicity of HIV-1 Infection among Injection Drug Users

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18-20, 43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (1, 13, 18, 24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15, 36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (19). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (8). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (1, 13, 18, 24). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (1, 13, 18, 24). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (1, 13, 18, 20, 24).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort (41)—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, 40, 41). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (4, 5, 9, 22, 23).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage (8) and to estimate the date of infection (Table ​(Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. ​(Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table ​(Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (1, 13, 18, 24) and mathematical modeling (18, 20).Open in a separate windowFIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (18, 20, 24, 44). The horizontal scale bars represent genetic distance. nt, nucleotide.

TABLE 1.

Subject demographics and HIV-1 envelope analysis results

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)