Strain CH3 Research Articles

Riemerella anatipestifer UvrC is required for iron utilization, biofilm formation and virulence

ABSTRACT UvrC is a subunit of excinuclease ABC, which mediates nucleotide excision repair (NER) in bacteria. Our previous studies showed that transposon Tn4531 insertion in the UvrC encoding gene Riean_1413 results in reduced biofilm formation by Riemerella anatipestifer strain CH3 and attenuates virulence of strain YZb1. In this study, whether R. anatipestifer UvrC has some biological functions other than NER was investigated. Firstly, the uvrC of R. anatipestifer strain Yb2 was in-frame deleted by homologous recombination, generating deletion mutant ΔuvrC, and its complemented strain cΔuvrC was constructed based on Escherichia coli – R. anatipestifer shuttle plasmid pRES. Compared to the wild-type (WT) R. anatipestifer strain Yb2, uvrC deleted mutant ΔuvrC significantly reduced biofilm formation, tolerance to H2O2- and HOCl-induced oxidative stress, iron utilization, and adhesion to and invasion of duck embryonic hepatocytes, but not its growth curve and proteolytic activity. In addition, animal experiments showed that the LD50 value of ΔuvrC in ducklings was about 13-fold higher than that of the WT, and the bacterial loads in ΔuvrC infected ducklings were significantly lower than those in Yb2-infected ducklings, indicating uvrC deletion in R. anatipestifer attenuated virulence. Taken together, the results of this study indicate that R. anatipestifer UvrC is required for iron utilization, biofilm formation, oxidative stress tolerance and virulence of strain Yb2, demonstrating multiple functions of UvrC. RESEARCH HIGHLIGHTS Deletion of uvrC in R. anatipestfer Yb2 significantly reduced its biofilm formation. uvrC deletion led to reduced tolerance to H2O2- and HOCl-induced oxidative stress. The iron utilization of uvrC deleted mutant was significantly reduced. The uvrC deletion in R. anatipestifer Yb2 attenuated its virulence.

Antimicrobial, anti-biofilm, antioxidant and cytotoxic effects of bacteriocin by Lactococcus lactis strain CH3 isolated from fermented dairy products—An in vitro and in silico approach

The current study aimed to screen bacteriocin producing LAB from different dairy products and evaluation of their biological properties. Initially, 12 (4-chess, 4-curd, and 4-yohurt) LAB species were isolated and only 4 isolates alone were selected based on their clear yellow halo zone around the colonies in the selective medium. The selected 4 isolates were identified based on their morphological and biochemical characteristics. Among them, the strain CH3 have showed better antimicrobial effects on selected human pathogens. The isolated strain CH3 were further identified as Lactococcus lactis strain CH3 (MZ636710) by SEM imaging and 16 s rRNA molecular sequencing. Bacteriocin was extracted from L. lactis strain CH3 and partially purified using 60 % ammonium sulphate and then completely purified by G-50 column chromatography. The purified bacteriocin showed a specific activity of 5859.37 AU/mg in 24.7 % of recovery and 10.9-fold purification. The molecular weight of bacteriocin was 3.5 kDa as observed in SDS-PAGE. The bacteriocin showed sensitivity to proteolytic enzymes and resistance to high temperature, wide range of pH, organic solvents and detergents. FT-IR spectral studies of bacteriocin detected the existence of OH/NH-stretching, CH, and COC and CO bonds. NMR spectrum showed one doublet and 4 various singlet peaks at different ppm, indicating the occurrence of six amino acids in the structure of purified bacteriocin. The purified bacteriocin have shown stronger antimicrobial and anti-biofilm activity against selected human pathogens at 100 μg/mL. SEM showed the evidence of structural deformation and loss of membrane integrity of bacterial cells treated with bacteriocin. Bacteriocin exhibited greater DPPH radical scavenging potential with an EC50 value of 12.5 μg/mL. Bacteriocin have not shown significant toxicity on normal human dermal fibroblast (NHDF) cells (83.2 % at 100 μg/ mL). Furthermore, in silico studies using molecular modeling and docking were performed to know the proteins involved in antimicrobial action. The results suggests that bacteriocin could be an alternative to combat AMR pathogens and more suitable for food and dairy industries to preserve food without contamination.

Effective degradation of Chlortetracycline using dual bio catalyst

Chlortetracycline (CTC) degradation using potential microbial consortia or individual bacterial strains was useful method for improving bioremediation potential. The co-culture (Klebsiella pneumoniae CH3 and Bacillus amyloliquefaciens CS1) of bacterial strains have the ability to degrade chlortetracycline (91.8 ± 1.7%), followed by sulfamethoxazole (62.1 ± 1.2%) and amoxicillin (73.9 ± 3.3%). It was observed that the degradation potential was maximum after 10 days incubation, 8–10% inoculum, pH 7.5, and antibiotic concentration ranged from 150 to 200 mg/L. The initial concentrations of CTC significantly affected CTC degradation. In strain CH3, maximum biodegradation of CTC (99.4 ± 2.3%) was observed at 200 mg/L initial CTC concentrations. In CS1, maximum biodegradation of CTC was obtained at 150 mg/L concentration (80.5 ± 3.2%) after 10 days of culture. Alkaline pH was found to be suitable for the degradation of antibiotic than acidic range. After initial optimization by one factor at a time approach in free cells, the bacterial strains (CH3 and CS1) were co-immobilized. The co-immobilized bacterial cells showed improved degradation potential than free cells. To determine the biodegradation potential of immobilized cells, the selected strains were immobilized in polymer beads and treated with CTC with 175 mg/L initial concentration. The experimental results revealed that after 3 days of treatment the residual CTC concentration was 150.1 ± 3.2 mg/L and it decreased as 1.28 ± 0.01 mg/L after 10 days of treatment. The present study confirmed the effectiveness and feasibility of biodegradation ability of K. pneumoniae CH3 and B. amyloliquefaciens CS1 immobilized for CTC degradation in wastewater.

Characterization of the Riemerella anatipestifer M949_RS00050 gene

Based on its causing ever-increasing heavy economic losses, Riemerella anatipestifer has been viewed as an important bacterial pathogen in the duck industry worldwide. However, the molecular mechanisms regarding its pathogenicity are poorly understood. In our previous study, we have built a random mutagenesis library of Riemerella anatipestifer CH3 using transposon Tn4351. In this study, we screened the library by determining bacterial median lethal dose in ducklings. A mutant strain showed about 376-fold attenuated virulence in comparison with the wild-type strain CH3 was obtained. Subsequently, the Tn4351 inserted gene was identified as M949_RS00050, which encodes a putative protein containing an outer membrane protein beta-barrel domain by genome walking and sequence analyses. Southern blot analysis indicated a single Tn4351 insertion in the CH3 chromosomal DNA. Inactivation of M949_RS00050 gene did not affect bacterial metabolic activity and the silver stained lipopolysaccharide pattern. However, the bacterial sensitivity to normal duck sera killing and bacterial hydrophobicity were dramatically enhanced in the M949_RS00050 gene inactivated mutant strain, compared to its wild-type strain CH3. Moreover, bacterial adherence and invasion abilities, bacterial capsular polysaccharide quantity, biofilm formation capacity and the bacterial virulence of the mutant strain were obviously decreased, compared to the wild-type strain CH3. Thus, our finding demonstrates that the M949_RS00050 gene functions on multiple bacterial biological properties and virulence in Riemerella anatipestifer.

The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.

Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline.

Based on its important role in last-line therapeutics against multidrug-resistant bacteria, tigecycline has been increasingly important in treating infections. However, mounting reports on tigecycline-resistant bacterial strains isolated from different sources are of concern, and molecular mechanisms regarding tigecycline resistance are poorly understood. Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes fibrinous pericarditis, perihepatitis, and meningitis in infected ducks. We previously constructed a random transposon mutant library using Riemerella anatipestifer strain CH3, in present study, we described that Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline. Using the minimum inhibitory concentration assay, a mutant strain showed significantly increased (about six-fold) tigecycline susceptibility. Subsequently, the knocked-down gene was identified as M949_0459, a putative flavin adenine dinucleotide-dependent oxidoreductase. To confirm the resistance function, M949_0459 gene was overexpressed in Escherichia coli strain BL21, and the minimum inhibitory concentration analysis showed that the gene product conferred resistance to tigecycline. Additionally, expression of the M949_0459 gene under treatment with tigecycline was measured with quantitative real-time PCR. Results showed that the mRNA expression of M949_0459 gene was elevated under tigecycline treatment with dose range of 1-10 mg/L, and peaked at 4 mg/L. Moreover, two kinds of efflux pump inhibitors, carbonyl cyanide m-chlorophenyl hydrazone and phenylalanine arginyl β-naphthylamide were tested, which showed no function on tigecycline resistance in the strain CH3. Our results may provide insights into molecular mechanisms for chemotherapy in combating Riemerella anatipestifer infections.

Disruption of the M949_RS01915 gene changed the bacterial lipopolysaccharide pattern, pathogenicity and gene expression of Riemerella anatipestifer

Riemerella anatipestifer is an important pathogen that causes septicemia anserum exsudativa in ducks. Lipopolysaccharide (LPS) is considered to be a major virulence factor of R. anatipestifer. To identify genes involved in LPS biosynthesis, we screened a library of random Tn4351 transposon mutants using a monoclonal antibody against R. anatipestifer serotype 1 LPS (anti-LPS MAb). A mutant strain RA1067 which lost the reactivity in an indirect ELISA was obtained. Southern blot and sequencing analyses indicated a single Tn4351 was inserted at 116 bp in the M949_RS01915 gene in the RA1067 chromosomal DNA. Silver staining and Western blot analyses indicated that the RA1067 LPS was defected compared to the wild-type strain CH3 LPS. The RA1067 displayed a significant decreased growth rate at the late stage of growth in TSB in comparison with CH3. In addition, RA1067 showed higher susceptibility to complement-dependent killing, more than 360-fold attenuated virulence based on the median lethal dose determination, increased bacterial adhesion and invasion capacities to Vero cells and significantly decreased blood bacterial loads in RA1067 infected ducks, when compared to the CH3. An animal experiment indicated that inactivated RA1067 cells was effective in cross-protecting of the ducks from challenging with R. anatipestifer strains WJ4 (serotype 1), Yb2 (serotype 2) and HXb2 (serotype 10), further confirming the alteration of the RA1067 antigenicity. Moreover, RNA-Seq analysis and real-time PCR verified two up-regulated and three down-regulated genes in RA1067. Our findings demonstrate that the M949_RS01915 gene is associated to bacterial antigenicity, pathogenicity and gene regulation of R. anatipestifer.

Riemerella anatipestifer M949_1360 Gene Functions on the Lipopolysaccharide Biosynthesis and Bacterial Virulence

Riemerella anatipestifer causes septicemic and exudative diseases in poultry, resulting in major economic losses to the duck industry. Lipopolysaccharide (LPS), as an important virulence factor in Gram-negative bacteria, can be recognized by the immune system and plays a crucial role in many interactions between bacteria and animal hosts. In this study, we screened out one LPS defective mutant strain RAΔ604 from a random transposon mutant library of R. anatipestifer serotype 1 strain CH3, which did not react with the anti-CH3 LPS monoclonal antibody 1C1 in an indirect enzyme-linked immunosorbent assay. Southern blot analysis confirmed that the genome of RAΔ604 contained a single Tn4351 insert. Then, we found that the M949_1360 gene was inactivated by insertion of the transposon. Using silver staining and western blot analyses, we found that the LPS pattern of RAΔ604 was defective, as compared with that of the wild-type (WT) strain CH3. The mutant strain RAΔ604 showed no significant influence on bacterial growth, while bacterial counting and Live/dead BacLight Bacterial Viability staining revealed that bacterial viability was decreased, as compared with the WT strain CH3. In addition, the abilities of the mutant strain RAΔ604 to adhere and invade Vero cells were significantly decreased. Animal studies revealed that the virulence of the mutant strain RAΔ604 was decreased by more than 200-fold in a duck infection model, as compared with the WT strain CH3. Furthermore, immunization with live bacteria of the mutant strain RAΔ604 protected 87.5% ducks from challenge with R. anatipestifer serotype 1 strain WJ4, indicating that the mutant strain RAΔ604 could be used as a potential vaccine candidate in the future.

Characterization and cross-protection evaluation of M949_1603 gene deletion Riemerella anatipestifer mutant RA-M1.

Riemerella anatipestifer infection causes high mortality for ducks which results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn4351 transposon mutagenesis, in which the M949_1603 gene encoding glycosyl transferase was inactivated. PCR analysis revealed that M949_1603 gene is specifically existed in R. anatipestifer serotype 1 strains. RA-M1 presented no reactivity to the anti-lipopolysaccharide (LPS) MAb in an indirect ELISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting demonstrated that RA-M1 LPS had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of RA-M1 was changed. RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, cross-protection of RA-M1 for R. anatipestifer serotypes 1, 2, and 10 strains was evaluated. Ducks that received two immunizations with inactivated RA-M1 vaccine were 100% protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2, and serotype 10 strain HXb2. No changes were observed in the liver, heart, or spleen samples from the protected ducks during autopsy and histological examination. Furthermore, vaccination generated high antibody titers of 1:12,800 against serotypes 1, 2, and 10 strains and enhanced production of interleukin 2 (IL-2) and IL-4 in ducks. These results suggested that M949_1603 gene is associated with serotype 1 O-antigen biosynthesis, and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.

Roles of the TonB1 and TonB2 proteins in haemin iron acquisition and virulence in Riemerella anatipestifer.

Two TonB systems in Riemerella anatipestifer were found and characterized as ExbB1-ExbD1-TonB1 and ExbB2-ExbD2-ExbD2'-TonB2, but the significance of two sets of TonB complexes in R. anatipestifer is not clear. In this study, by deleting the tonB1 or tonB2 gene of R. anatipestifer strain CH3, we investigated the roles of the TonB1 and TonB2 proteins in iron acquisition and virulence. The results showed that strain CH3 could utilize haemin as the sole iron source in the presence of l-cysteine, but haemin iron acquisition was defective in the CH3ΔtonB1 mutant, and the deletion of either tonB1 or tonB2 significantly reduced adhesion to and invasion of Vero cells. Animal experiments indicated that the LD50 of the CH3ΔtonB1 and CH3ΔtonB2 mutants in ducklings was ∼224- and ∼87-fold, respectively, higher than that of the WT CH3 strain. Additional analysis indicated that blood bacterial loading of ducklings infected with CH3ΔtonB1 or CH3ΔtonB2 decreased significantly compared with that found for WT CH3-infected ducklings. Thus, our results indicated that the TonB1, but not TonB2 protein, is involved in haemin iron acquisition and that both TonB proteins are necessary for optimal bacterial virulence.

The M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of Riemerella anatipestifer

Riemerella anatipestifer is one of the most economically important pathogens of farm ducks worldwide. However, the molecular mechanisms regarding its antigenicity and pathogenicity are poorly understood. We previously constructed a library of random Tn4351 transposon mutants using R. anatipestifer strain CH3. In this study, M949_1556 gene inactivated mutant strain CH3ΔM949_1556 was identified by screening of the library using monoclonal antibody against R. anatipestifer serotype 1 lipopolysaccharide (LPS) (anti-LPS MAb) followed by sequence analysis. The mutant strain presented no reactivity to the anti-LPS MAb in an indirect ELISA. Animal studies showed that the median lethal dose (LD50) of CH3ΔM949_1556 was >1010 colony forming units (CFU), which was attenuated more than 50 times, compared with that of wild-type strain CH3 (LD50=2×108CFU). The bacterial loads in the blood of CH3ΔM949_1556 infected ducks were significantly decreased, compared with those of CH3-infected ducks. In addition, CH3ΔM949_1556 presented significant, higher susceptibility to complement-dependent killing than CH3 did in vitro. Furthermore, CH3ΔM949_1556 showed increased bacterial adhesion and invasion capacities to Vero cells. Immunization with CH3ΔM949_1556-inactived vaccine was effective in protecting the ducks from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2 and serotype 10 strain HXb2, suggesting that the mutant strain CH3ΔM949_1556 could provide a broad cross-protection among R. anatipestifer serotypes 1, 2 and 10 strains. Our results demonstrated that the M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of R. anatipestifer.

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.

The role of TonB-dependent receptor TbdR1 in Riemerella anatipestifer in iron acquisition and virulence

Riemerella anatipestifer is an important duck pathogen and causes serious economic losses to the duck industry worldwide. To date, four full R. anatipestifer genomic sequences have been submitted to the GenBank database and 31 TonB-dependent outer membrane receptors, which may play critical roles in host–bacteria interactions, were predicted for R. anatipestifer strain GSM15868. In our previous study, we reported that the TonB-dependent receptor TbdR1 was a cross immunogenic antigen among R. anatipestifer serotypes 1, 2, and 10. However, the biological functions of TbdR1 in R. anatipestifer remain unclear. In the present study, a tbdR1 (Riean_1607) deletion mutant CH3ΔtbdR1 of R. anatipestifer strain CH3 was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Our results showed that TbdR1 was involved in hemin iron acquisition and the tbdR1 deletion significantly reduced biofilm formation and adhesion to and invasion of Vero cells. Animal experiments indicated that the median lethal dose of the CH3ΔtbdR1 mutant in ducklings was about 45-fold higher than that of the wild-type CH3 strain. Additional analysis indicated that bacterial loads in blood, liver, and brain tissues in CH3ΔtbdR1-infected ducklings were decreased significantly compared to those in wild-type CH3-infected ducklings. Thus, our results demonstrated that TbdR1 was involved in hemin iron acquisition and necessary for optimal bacterial virulence.

Identification of the Genes Involved in Riemerella anatipestifer Biofilm Formation by Random Transposon Mutagenesis

Riemerella anatipestifer causes epizootics of infectious disease in poultry that result in serious economic losses to the duck industry. Our previous studies have shown that some strains of R. anatipestifer can form a biofilm, and this may explain the intriguing persistence of R. anatipestifer on duck farms post infection. In this study we used strain CH3, a strong producer of biofilm, to construct a library of random Tn4351 transposon mutants in order to investigate the genetic basis of biofilm formation by R. anatipestifer on abiotic surfaces. A total of 2,520 mutants were obtained and 39 of them showed a reduction in biofilm formation of 47%–98% using crystal violet staining. Genetic characterization of the mutants led to the identification of 33 genes. Of these, 29 genes are associated with information storage and processing, as well as basic cellular processes and metabolism; the function of the other four genes is currently unknown. In addition, a mutant strain BF19, in which biofilm formation was reduced by 98% following insertion of the Tn4351 transposon at the dihydrodipicolinate synthase (dhdps) gene, was complemented with a shuttle plasmid pCP-dhdps. The complemented mutant strain was restored to give 92.6% of the biofilm formation of the wild-type strain CH3, which indicates that the dhdp gene is associated with biofilm formation. It is inferred that such complementation applies also to other mutant strains. Furthermore, some biological characteristics of biofilm-defective mutants were investigated, indicating that the genes deleted in the mutant strains function in the biofilm formation of R. anatipestifer. Deletion of either gene will stall the biofilm formation at a specific stage thus preventing further biofilm development. In addition, the tested biofilm-defective mutants had different adherence capacity to Vero cells. This study will help us to understand the molecular mechanisms of biofilm development by R. anatipestifer and to study the pathogenesis of R. anatipestifer further.

케라틴 단백질 폐기물의 재활용을 위한 우모부해 세균의 분리와 특성

The aim of this study was to isolate chicken feather-degrading bacteria with high keratinolytic activity and to investigate cultural conditions affecting keratinolytic enzyme production by a selected isolate. A chicken feather-degrading bacterial strain CH3 was isolated from poultry wastes. Isolate CH3 degraded whole chicken feather completely within 3 days. On the basis of phenotypical and 16S rDNA studies, isolate CH3 was identified as Bacillus thuringiensis CH3. This strain is the first B. thuringiensis described as a feather degrader. The bacterium grew with an optimum at pH 8.0 and <TEX>$37^{\circ}C$</TEX>, where maximum keratinolytic activity was also observed. The composition of optimal medium for keratinolytic enzyme production was feather 0.1%, sucrose 0.7%, casein 0.3%, <TEX>$K_2HPO_4$</TEX> 0.03%, <TEX>$KH_2PO_4$</TEX> 0.04%, <TEX>$MgCl_2$</TEX> 0.01% and NaCl 0.05%, respectively. The keratinolytic enzyme had a pH and temperature optima 9.0 and <TEX>$45^{\circ}C$</TEX>, respectively. The keratinolytic activity was inhibited ethylenediaminetetraacetic acid, phenylmethylsulfonyl fluoride, and metal ions like <TEX>$Hg^{2+},\;Cu^{2+}\;and\;Zn^{2+}$</TEX>. The enzyme activated by <TEX>$Fe^{2+}$</TEX>, dithiothreitol and 2-mercaptoethanol.

Effect of growth conditions on the hydrogen production with cyanobacterium Anabaena sp. strain CH3

Cyanobacteria could use sugars as carbon source and reductant to produce hydrogen by nitrogenase. However, oxygen is also produced during photosynthesis and it is an inhibitor of the enzyme nitrogenase. Filamentous cyanobacterium Anabaena sp. CH 3 could use sugars as substrate to produce molecular hydrogen anaerobically. The production activity was dependent on growth phases. It was found that the cells at sub-stage of late-log phase had better ability to produce hydrogen than at log phase. In such case, oxygen content was too low to be detected to inhibit hydrogen production. Among different kinds of sugar, fructose and glucose had the best performance for producing hydrogen. Hydrogen could be accumulated to 0.6 mmol (in 40 ml head space) in 100 h from 1000 ppm fructose. Increasing light intensities from 65 to 130 μ mol m - 2 s - 1 would enhance hydrogen production to 0.8 mmol. Under illumination of 130 μ mol m - 2 s - 1 and 2000 ppm fructose, 1.7 mmol of hydrogen could be accumulated. When fructose content was higher than 2000 ppm, cells could not produce more hydrogen at all.

Study of Protection by Recombinant Fowl Poxvirus Expressing C-Terminal Nucleocapsid Protein of Infectious Bronchitis Virus against Challenge

A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains.

A minor prevalent strain in a severe outbreak of foal diarrhea associated with serotype 3 rotavirus.

An epizootic of foal diarrhea due to serotype 3 rotavirus (RV) was observed in 89 of 168 cases (53%) during the period from March to July in 1987. A total of 51 strains of RV were isolated from the 62 diarrheal feces examined, and one isolate (CH-3) showed a unique electropherotype of viral RNA which differed from the others that widely prevailed on this farm. No positive reaction was observed between strain CH-3 and each of the antisera against serotypes 1 to 12 of human and animal RV in neutralization tests. However, dsRNAs of the CH-3 virus were hybridized with a probe prepared from a strain of equine serotype 3 RV.