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Nitrate respiration is a widespread mode of anaerobic energy generation used by many bacterial pathogens, and the respiratory nitrate reductase, Nar, has long been known to reduce chlorate to the toxic oxidizing agent chlorite. Here, we demonstrate the antibacterial activity of chlorate against Pseudomonas aeruginosa, a representative pathogen that can inhabit hypoxic or anoxic host microenvironments during infection. Aerobically grown P. aeruginosa cells are tobramycin sensitive but chlorate tolerant. In the absence of oxygen or an alternative electron acceptor, cells are tobramycin tolerant but chlorate sensitive via Nar-dependent reduction. The fact that chlorite, the product of chlorate reduction, is not detected in culture supernatants suggests that it may react rapidly and be retained intracellularly. Tobramycin and chlorate target distinct populations within metabolically stratified aggregate biofilms; tobramycin kills cells on the oxic periphery, whereas chlorate kills hypoxic and anoxic cells in the interior. In a matrix populated by multiple aggregates, tobramycin-mediated death of surface aggregates enables deeper oxygen penetration into the matrix, benefiting select aggregate populations by increasing survival and removing chlorate sensitivity. Finally, lasR mutants, which commonly arise in P. aeruginosa infections and are known to withstand conventional antibiotic treatment, are hypersensitive to chlorate. A lasR mutant shows a propensity to respire nitrate and reduce chlorate more rapidly than the wild type does, consistent with its heightened chlorate sensitivity. These findings illustrate chlorate’s potential to selectively target oxidant-starved pathogens, including physiological states and genotypes of P. aeruginosa that represent antibiotic-tolerant populations during infections. IMPORTANCE The anaerobic growth and survival of bacteria are often correlated with physiological tolerance to conventional antibiotics, motivating the development of novel strategies targeting pathogens in anoxic environments. A key challenge is to identify drug targets that are specific to this metabolic state. Chlorate is a nontoxic compound that can be reduced to toxic chlorite by a widespread enzyme of anaerobic metabolism. We tested the antibacterial properties of chlorate against Pseudomonas aeruginosa, a pathogen that can inhabit hypoxic or anoxic microenvironments, including those that arise in human infection. Chlorate and the antibiotic tobramycin kill distinct metabolic populations in P. aeruginosa biofilms, where chlorate targets anaerobic cells that tolerate tobramycin. Chlorate is particularly effective against P. aeruginosa lasR mutants, which are frequently isolated from human infections and more resistant to some antibiotics. This work suggests that chlorate may hold potential as an anaerobic prodrug.

Supplemental material for this article may be found at https://doi.org/10.1128/AEM .00325-17., Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1T, a facultative anaerobic chloratereducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the L-DEX gene and dehI, encoding an L-2-haloacid dehalogenase (L-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1T. The deduced amino acid sequences of L-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized L-DEX and the D- and DL-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1T can grow on chloroacetate, bromoacetate, and both L- and D--halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of L-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1T grown on DL-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1T cells grown on DL-2-chloropropionate under aerobic conditions. Reverse transcriptionquantitative PCR showed that the L-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle. © 2017 American Society for Microbiology., This project is financially supported by the BE-Basic Foundation through project MicroControl (8.004.01). P.P. and Y.Z. are sponsored by China Scholarship Council. The research of A.J.M.S. is supported by an ERC grant (project 323009) and the gravitation grant (project 024.002.002) of The Netherlands Ministry of Education, Culture and Science., info:eu-repo/semantics/publishedVersion

The ability of microorganisms to perform dissimilatory (per)chlorate reduction is, for most species, known to be oxygen sensitive. Consequently, bioremediation processes for the removal of oxochlorates will be disturbed if oxygen is present. We measured the expression of chlorite dismutase and chlorate reductase in the presence of different terminal electron acceptors in the chlorate reducer Ideonella dechloratans . Enzyme activity assays and mRNA analyses by real-time quantitative reverse transcription (qRT)-PCR were performed on cell extracts from cells grown aerobically with and without chlorate and on cells grown anaerobically in the presence of chlorate. Our results showed that both chlorite dismutase and chlorate reductase are expressed during aerobic growth. However, transfer to anaerobic conditions with chlorate resulted in significantly enhanced enzyme activities and mRNA levels for both enzymes. Absence of oxygen was necessary for the induction to occur, since chlorate addition under aerobic conditions produced neither increased enzyme activities nor higher relative levels of mRNA. For chlorite dismutase, the observed increase in activity was on the same order of magnitude as the increase in the relative mRNA level, indicating gene regulation at the transcriptional level. However, chlorate reductase showed about 200 times higher enzyme activity in anaerobically induced cells, whereas the increase in mRNA was only about 10-fold, suggesting additional mechanisms influence the enzyme activity.

The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c -type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectroscopy to determine the ability of donating electrons to chlorate reductase. Our results show that one of the c cytochromes (6 kDa) is able to donate electrons, both to chlorate reductase and to the membrane-bound cytochrome c oxidase, whereas the roles of the remaining c cytochromes still remain to be elucidated. Peptide extracts of the c cytochromes were obtained by tryptic in-gel digestion for matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Peptide sequences obtained indicate that the 6-kDa cytochrome c protein is similar to c cytochromes from the chlorate-reducing bacterium Dechloromonas aromatica .

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase ( cld ) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.

It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F 1 and F 2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.

Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrP Sc . Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrP Sc uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrP Sc formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrP Sc formation independent of the prion strain. IMPORTANCE Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro . GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.

The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2−), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms., IMPORTANCE S. selenatireducens CUZ and D. chlorophilus NSS are (per)chlorate- and chlorate-reducing bacteria, respectively, whose genomes encode both anaerobic and aerobic-hybrid pathways for the degradation of phenylacetate and benzoate. Previous studies have shown that (per)chlorate-reducing bacteria and chlorate-reducing bacteria (CRB) can use aerobic pathways to oxidize aromatic compounds in otherwise anoxic environments by capturing the oxygen produced from chlorite dismutation. In contrast, we demonstrate that S. selenatireducens CUZ is the first perchlorate reducer known to utilize anaerobic aromatic degradation pathways with perchlorate as an electron acceptor and that it does so in preference over the aerobic-hybrid pathways, regardless of any oxygen produced from chlorite dismutation. D. chlorophilus NSS, on the other hand, may be carrying out anaerobic and aerobic-hybrid processes simultaneously. Concurrent use of anaerobic and aerobic pathways has not been previously reported for other CRB or any microorganisms that encode similar pathways of phenylacetate or benzoate degradation and may be advantageous in low-oxygen environments.

Haemophilus somnuscan cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used byH. somnusto migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate thatH. somnusadheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherentH. somnuswas significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-α). Addition of exogenous glycosaminoglycans significantly reducedH. somnusadherence to resting and TNF-α-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherentH. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence ofH. somnusto BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed byH. somnus. Heparin was also shown to bindH. somnusin a 4°C binding assay. These data suggest that heparin-binding proteins onH. somnuscould serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability ofH. somnusto infect the bovine CNS.

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10 −6 of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata , but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata , dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.

Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 × 10 3 to 2.4 × 10 6 cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 μmol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria . Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera Pseudomonas and Azospirllum . However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria . The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α 3 β 3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K m values for perchlorate and chlorate were 27 and

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA − phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.

Functional disruption of the gene encoding nitrate reductase (niaD) in Aspergillus parasiticus was conducted by two strategies, one-step gene replacement and the integrative disruption. Plasmid pPN-1, in which an internal DNA fragment of the niaD gene was replaced by a functional gene encoding orotidine monophosphate decarboxylase (pyrG), was constructed. Plasmid pPN-1 was introduced in linear form into A. parasiticus CS10 (ver-1 wh-1 pyrG) by transformation. Approximately 25% of the uridine prototrophic transformants (pyrG+) were chlorate resistant (Chlr), demonstrating their inability to utilize nitrate as a sole nitrogen source. The genetic block in nitrate utilization was confirmed to occur in the niaD gene by the absence of growth of the A. parasiticus CS10 transformants on medium containing nitrate as the sole nitrogen source and the ability to grow on several alternative nitrogen sources. Southern hybridization analysis of Chlr transformants demonstrated that the resident niaD locus was replaced by the nonfunctional allele in pPN-1. To generate an integrative disruption vector (pSKPYRG), an internal fragment of the niaD gene was subcloned into a plasmid containing the pyrG gene as a selectable marker. Circular pSKPYRG was transformed into A. parasiticus CS10. Chlr pyrG+ transformants were screened for nitrate utilization and by Southern hybridization analysis. Integrative disruption of the genomic niaD gene occurred in less than 2% of the transformants. Three gene replacement disruption transformants and two integrative disruption transformants were tested for mitotic stability after growth under nonselective conditions. All five transformants were found to stably retain the Chlr phenotype after growth on nonselective medium.(ABSTRACT TRUNCATED AT 250 WORDS)

All molybdoenzyme activities are absent in chlB mutants because of their inability to synthesize molybdopterin guanine dinucleotide, which together with molybdate constitutes the molybdenum cofactor in Escherichia coli. The chlB mutants are able to synthesize molybdopterin. We have previously shown that the inactive nitrate reductase present in a chlB mutant can be activated in a process requiring protein FA and a heat-stable low-molecular-weight substance. We show here that purified nitrate reductase from the soluble fraction of a chlB mutant can be partially activated in a process that requires protein FA, GTP, and an additional protein termed factor X. It appears that the molybdopterin present in the nitrate reductase of a chlB mutant is converted to molybdopterin guanine dinucleotide during activation. The activation is absolutely dependent upon both protein FA and factor X. Factor X activity is present in chlA, chlB, chlE, and chlG mutants.

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N -sulfation was three times less efficient than infection of the parental CHO cell line, indicating that N -sulfation is important. In contrast, infection of a cell line deficient in 2- O -sulfation was as efficient as infection of the parental cell line, indicating that 2- O -sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack their N -, C6- O -, or C2- O -sulfate groups. Only heparin chains lacking the N -sulfate group lost the ability to neutralize infection, confirming that N -sulfation, but not C6- O - or C2- O -sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes N -sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264–275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.

The cytochromes present in the membranes of Escherichia coli cells having defects in the formate dehydrogenase-nitrate reductase system have been analyzed by spectroscopic, redox titration, and enzyme fractionation techniques. Four phenotypic classes differing in cytochrome composition were recognized. Class I is represented by strains with defects in the synthesis or insertion of molybdenum cofactor. Cytochromes of the formate dehydrogenase-nitrate reductase pathway are present. Class II strains map in the chlC-chlI region. The cytochrome associated with nitrate reductase (cytochrome bnr) is absent in these strains, whereas that associated with formate dehydrogenase (cytochrome bfdh) is the major cytochrome in the membranes. Class III strains lack both cytochromes bfdh and bnr but overproduce cytochrome d of the aerobic pathway even under anaerobic conditions in the presence of nitrate. Class III strains have defects in the regulation of cytochrome synthesis. An fdhA mutant produced cytochrome bnr but lacked cytochrome bfdh. These results support the view that chlI (narI) is the structural gene for cytochrome bnr and that chlC (narG) and chlI(narI) are in the same operon, and they provide evidence of the complexity of the regulation of cytochrome synthesis.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 microM was estimated. In uptake assays, the crnA1 mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnA1 reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction.

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b -type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.

We have developed a convenient and specific positive selection for long deletions through the gal region of the chromosomes of Salmonella typhimurium and Escherichia coli. Through simultaneous selection for mutations in the two closely linked genes, gal and chlA, a variety of deletions of varying length, some extending through as much as 1 min of the chromosome, could be readily obtained. Many of these deletions resulted in the loss of a gene, which we named dhb, concerned with the ability of the bacterium to synthesize the iron chelating agent enterobactin. The selection was adapted for the screening of mutagens for their ability to generate long deletions in the bacterial deoxyribonucleic acid. Forty agents were screened for this capability. Nitrous acid, previously reported to be an efficient mutagen for this purpose, increased the frequency of deletion mutations 50-fold in our system. Three others, nitrogen mustard, mitomycin C, and fast neutrons, were shown to increase the frequency of long deletions between five- and eightfold. The remainder were found to be incapable of generating these deletions.

We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis of insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::Tn10 mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.

Acid-treated extracts of Escherichia coli were tested for their ability to restore reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity to an extract of a Neurospora nit-1 mutant which produces a defective enzyme. With wild-type E. coli this complementation activity was more readily detected in the cytoplasmic fraction, although the nitrate reductase activity was found primarily in the particulate fraction. chlB mutants of E. coli appeared to have more complementation activity in the cytoplasm than was observed in the wild type, but no activity in the particulate fraction. The other chl mutants had little or no activity in either fraction. These results suggest that chlB mutants can produce a component or cofactor which is missing in the other mutants and in the Neurospora mutant, but cannot transfer it to the nitrate reductase enzyme.

Chlorate-resistant mutants corresponding to each known genetic locus ( chlA, chlB, chlC, chlD, chlE ) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b , and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.

The bacterial virus f2 was inactivated by chlorine dioxide at acidic, neutral, and alkaline pH values. The rate of inactivation increased with increasing pH. Chlorine dioxide disproportionation products, chlorite and chlorate, were not active disinfectants. As chlorine dioxide solutions were degraded under alkaline conditions, they displayed reduced viricidal effectiveness, thereby confirming the chlorine dioxide free radical as the active disinfecting species.