Showing papers in "Journal of Microbiology in 2005"

The Viable but Nonculturable State in Bacteria

Abstract: It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

Quorum sensing and quorum-quenching enzymes.

Abstract: To gain maximal benefit in a competitive environment, single-celled bacteria have adopted a community genetic regulatory mechanism, known as quorum sensing (QS). Many bacteria use QS signaling systems to synchronize target gene expression and coordinate biological activities among a local population. N-acylhomoserine lactones (AHLs) are one family of the well-characterized QS signals in Gram-negative bacteria, which regulate a range of important biological functions, including virulence and biofilm formation. Several groups of AHL-degradation enzymes have recently been identified in a range of living organisms, including bacteria and eukaryotes. Expression of these enzymes in AHL-dependent pathogens and transgenic plants efficiently quenches the microbial QS signaling and blocks pathogenic infections. Discovery of these novel quorum quenching enzymes has not only provided a promising means to control bacterial infections, but also presents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems.

Laboratory diagnosis of invasive candidiasis.

Abstract: Invasive candidiasis is associated with high morbidity and mortality. Clinical diagnosis is complicated by a lack of specific clinical signs and symptoms of disease. Laboratory diagnosis is also complex because circulating antibodies to Candida species may occur in normal individuals as the result of commensal colonization of mucosal surfaces thereby reducing the usefulness of antibody detection for the diagnosis of this disease. In addition, Candida species antigens are often rapidly cleared from the circulation so that antigen detection tests often lack the desired level of sensitivity. Microbiological confirmation is difficult because blood cultures can be negative in up to 50% of autopsy-proven cases of deep-seated candidiasis or may only become positive late in the infection. Positive cultures from urine or mucosal surfaces do not necessarily indicate invasive disease although can occur during systemic infection. Furthermore, differences in the virulence and in the susceptibility of the various Candida species to antifungal drugs make identification to the species level important for clinical management. Newer molecular biological tests have generated interest but are not yet standardized or readily available in most clinical laboratory settings nor have they been validated in large clinical trials. Laboratory surveillance of at-risk patients could result in earlier initiation of antifungal therapy if sensitive and specific diagnostic tests, which are also cost effective, become available. This review will compare diagnostic tests currently in use as well as those under development by describing their assets and limitations for the diagnosis of invasive candidiasis.

Molecular Pathogenesis of Vibrio vulnificus.

Abstract: Vibrio vulnificus is an opportunistic pathogen of humans that has the capability of causing rare, yet devastating disease. The bacteria are naturally present in estuarine environments and frequently contaminate seafoods. Within days of consuming uncooked, contaminated seafood, predisposed individuals can succumb to sepsis. Additionally, in otherwise healthy people, V. vulnificus causes wound infection that can require amputation or lead to sepsis. These diseases share the characteristics that the bacteria multiply extremely rapidly in host tissues and cause extensive damage. Despite the analysis of virulence for over 20 years using a combination of animal and cell culture models, surprisingly little is known about the mechanisms by which V. vulnificus causes disease. This is in part because of differences observed using animal models that involve infection with bacteria versus injection of toxins. However, the increasing use of genetic analysis coupled with detailed animal models is revealing new insight into the pathogenesis of V. vulnificus disease.

Genetic and Environmental Control of Salmonella Invasion

Abstract: An early step in the pathogenesis of non-typhoidal Salmonella species is the ability to penetrate the intestinal epithelial monolayer. This process of cell invasion requires the production and transport of secreted effector proteins by a type III secretion apparatus encoded in Salmonella pathogenicity island I (SPI-1). The control of invasion involves a number of genetic regulators and environmental stimuli in complex relationships. SPI-1 itself encodes several transcriptional regulators (HilA, HilD, HilC, and InvF) with overlapping sets of target genes. These regulators are, in turn, controlled by both positive and regulators outside SPI-1, including the two-component regulators BarA/SirA and PhoP/Q, and the csr post-transcriptional control system. Additionally, several environmental conditions are known to regulate invasion, including pH, osmolarity, oxygen tension, bile, Mg2+ concentration, and short chain fatty acids. This review will discuss the current understanding of invasion control, with emphasis on the interaction of environmental factors with genetic regulators that leads to productive infection.

Sphingopyxis granuli sp. nov., a beta-glucosidase-producing bacterium in the family Sphingomonadaceae in alpha-4 subclass of the Proteobacteria.

Abstract: Strain Kw07T, a Gram-negative, non-spore-forming, rod-shaped bacterium, was isolated from granules in an Up-flow Anaerobic Sludge Blanket (UASB) bioreactor used in the treatment of brewery wastewater. 16S rRNA gene sequence analysis revealed that strain Kw07T belongs to the alpha-4 subclass of the Proteobacteria, and the highest degree of sequence similarity was determined to be to Sphingopyxis macrogoltabida IFO 15033T(97.8%). Chemotaxonomic data revealed that strain Kw07T possesses a quinone system with the predominant compound Q-10, the predominant fatty acid C18:1 omega7c, and sphingolipids, all of which corroborated our assignment of the strain to the Sphingopyxis genus. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that strain Kw07T represents a distinct species. Based on these data, Kw07T (=KCTC 12209T=NBRC 100800T) should be classified as the type strain for a novel Sphingopyxis species, for which the name Sphingopyxis granuli sp. nov. has been proposed.

Establishment of a micro-particle bombardment transformation system for Dunaliella salina.

Abstract: In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Gibberellins-producing rhizobacteria increase endogenous gibberellins content and promote growth of red peppers

Abstract: The growth of red pepper plants was enhanced by treatment with the rhizobacterium, Bacillus cereus MJ-1. Red pepper shoots showed a 1.38-fold increase in fresh weight (fw) and roots showed a 1.28-fold fw gain. This plant growth-promoting rhizobacterium (PGPR) has been reported to produce gibberellins (GAs). Other GAs-producing rhizobacteria, Bacillus macroides CJ-29 and Bacillus pumilus CJ-69, also enhanced the fw of the plants. They were less effective than B. cereus MJ-1, though. The endogenous GAs content of pepper shoots inoculated with MJ-1 was also higher than in shoots inoculated with CJ-29 or CJ-69. When inoculated with MJ-1, bacterial colonization rate of the roots was higher than that of roots inoculated with CJ-29 or CJ-69. These results support the idea that the plant growth-promoting effect of the bacteria also positively related with the efficiency of root colonization by the bacteria. In addition, we identified the major endogenous GAs of the red pepper as originating from both the early C-13 hydroxylation and the early non C-13 hydroxylation pathways, with the latter being the predominant pathway of GA biosynthesis in red pepper shoots.

Purification and characterization of laccase from the white rot fungus Trametes versicolor.

Abstract: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

Microbial conversion of major ginsenoside rb(1) to pharmaceutically active minor ginsenoside rd.

Abstract: More than seventy strains of aerobic bacteria showing beta-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar, and identified by their 16S rRNA gene sequences. Of these microorganisms, twelve strains could convert the major ginsenoside, Rb(1), to the pharmaceutically active minor ginsenoside Rd. Three strains, Burkholderia pyrrocinia GP16, Bacillus megaterium GP27 and Sphingomonas echinoides GP50, were phylogenetically studied, and observed to be most potent at converting ginsenoside Rb(1) almost completely within 48 h, as shown by TLC and HPLC analyses.

Hepatitis C virus non-structural protein NS4B can modulate an unfolded protein response.

Abstract: Viral infection causes stress to the endoplasmic reticulum (ER) The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover The role of hepatitis C virus (HCV) non-structural protein NS4B, a component of the HCV replicons that induce UPR, is incompletely understood We demonstrate that HCV NS4B could induce activating transcription factor (ATF6) and inositol-requiring enzyme 1 (IRE1), to favor the HCV subreplicon and HCV viral replication HCV NS4B activated the IRE1 pathway, as indicated by splicing of X box-binding protein (Xbp-1) mRNA However, transcriptional activation of the XBP-1 target gene, EDEM (ER degradation-enhancing alpha-mannosidase-like protein, a protein degradation factor), was inhibited These results imply that NS4B might induce UPR through ATF6 and IRE1-XBP1 pathways, but might also modify the outcome to benefit HCV or HCV subreplicon replication

Molecular analysis of colonized bacteria in a human newborn infant gut.

Abstract: The complex ecosystem of intestinal microflora is estimated to harbor approximately 400 different microbial species, mostly bacteria. However, studies on bacterial colonization have mostly been based on culturing methods, which only detect a small fraction of the whole microbiotic ecosystem of the gut. To clarify the initial acquisition and subsequent colonization of bacteria in an infant within the few days after birth, phylogenetic analysis was performed using 16S rDNA sequences from the DNA isolated from feces on the 1st, 3rd, and 6th day. 16S rDNA libraries were constructed with the amplicons of PCR conditions at 30 cycles and 50 degrees c annealing temperature. Nine independent libraries were produced by the application of three sets of primers (set A, set B, and set C) combined with three fecal samples for day 1, day 3, and day 6 of life. Approximately 220 clones (76.7%) of all 325 isolated clones were characterized as known species, while other 105 clones (32.3%) were characterized as unknown species. The library clone with set A universal primers amplifying 350 bp displayed increased diversity by days. Thus, set A primers were better suited for this type of molecular ecological analysis. On the first day of the life of the infant, Enterobacter, Lactococcus lactis, Leuconostoc citreum, and Streptococcus mitis were present. The largest taxonomic group was L. lactis. On the third day of the life of the infant, Enterobacter, Enterococcus faecalis, Escherichia coli, S. mitis, and Streptococcus salivarius were present. On the sixth day of the life of the infant, Citrobacter, Clostridium difficile, Enterobacter sp., Enterobacter cloacae, and E. coli were present. The largest taxonomic group was E. coli. These results showed that microbiotic diversity changes very rapidly in the few days after birth, and the acquisition of unculturable bacteria expanded rapidly after the third day.

Isolation, Characterization, and Investigation of Surface and Hemolytic Activities of a Lipopeptide Biosurfactant Produced by Bacillus subtilis ATCC 6633

Abstract: Bacillus subtilis ATCC 6633 was grown in BHIB medium supplemented with Mn2+ for 96 h at 37 degrees C in a shaker incubator. After removing the microbial biomass, a lipopeptide biosurfactant was extracted from the supernatant. Its structure was established by chemical and spectroscopy methods. The structure was confirmed by physical properties, such as Hydrophile-Lipophile Balance (HLB), surface activity and erythrocyte hemolytic capacity. The critical micelle concentration (cmc) and erythrocyte hemolytic capacity of the biosurfactant were compared to those of surfactants such as SDS, BC (benzalkonium chloride), TTAB (tetradecyltrimethylammonium bromide) and HTAB (hexadecyltrimethylammonium bromide). The maximum hemolytic effect for all surfactants mentioned was observed at concentrations above cmc. The maximum hemolytic effect of synthetic surfactants was more than that of the biosurfactant produced by B. subtilis ATCC 6633. Therefore, biosurfactant would be considered a suitable surface-active agent due to low toxicity to the membrane.

Salmonella invasion gene regulation: a story of environmental awareness.

Abstract: Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. A critical virulence determinant of Salmonella is the ability to invade mammalian cells. The expression of genes required for invasion is tightly regulated by environmental conditions and a variety of regulatory genes. The hilA regulator encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. Work from several laboratories has highlighted that regulation of hilA expression is a key point for controlling expression of the invasive phenotype. A number of positive regulators of hilA expression have been identified including csrAB, sirA/barA, pstS, hilC/sirC/sprA, fis, and hilD. HilD, an AraC/XylS type transcriptional regulator, is of particular importance as a mutation in hilD results in a 14-fold decrease in chromosomal hilA::Tn5lacZY-080 expression and a 53-fold decrease in invasion of HEp-2 cells. It is believed that HilD directly regulates hilA expression as it has been shown to bind to hilA promoter sequences. In addition, our research group, and others, have identified genes (hilE, hha, pag, and lon) that negatively affect hilA transcription. HilE appears to be an important Salmonella-specific regulator that plays a critical role in inactivating hilA expression. Recent work in our lab has been directed at understanding how environmental signals that affect hilA expression may be processed through a hilE pathway to modulate expression of hilA and the invasive phenotype. The current understanding of this complex regulatory system is reviewed.

Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456.

Abstract: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.

Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients.

Abstract: The objective of this study was to detect and compare the presence of periodontopathogens in the subgingival plaques of gingivitis lesions in adults who wore fixed orthodontic appliances, as opposed to adults who did not wear any orthodontic appliances. Thirty-six individuals participated in this study. Nineteen of these subjects did not wear any orthodontic appliances, and these subjects comprised the control group. The other 17 individuals had been wearing fixed orthodontic appliances for at least 3 months each. After a periodontal examination, we collected subgingival plaque samples from the gingivitis lesions of each patient. Using PCR based on 16S rDNA, we detected the presence of 6 putative periodontopathogenic species, Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia (formerly Bacteroides forsythus), Prevotella nigrescens, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. With regard to the presence of individual periodontopathogens, we found that T. forsythia, T. denticola, and P. nigrescens were significantly more common in the samples obtained from the orthodontic patients than in the samples obtained from the non-orthodontic patient controls. Our results indicate that the local changes associated with the wearing of fixed orthodontic appliances may affect the prevalence of periodontopathogens in subgingival dental plaques.

Archaeal diversity in tidal flat sediment as revealed by 16S rDNA analysis.

Abstract: During the past ten years, Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages. More recently, the presence of novel archaeal phylogenetic lineages has been discovered in coastal marine environments, freshwater lakes, polar seas, and deep-sea hydrothermal vents. Therefore, we conducted an investigation into the archaeal community existing in tidal flat sediment collected from Ganghwa Island, Korea. Phylogenetic analysis of archaeal 16S rDNA amplified directly from tidal flat sediment DNA revealed the presence of two major lineages, belonging to the Crenarchaeota (53.9%) and Euryarchaeota (46.1%) phyla. A total of 102 clones were then sequenced and analyzed by comprehensive phylogenetic analysis. The sequences determined in our samples were found to be closely related to the sequences of clones which had been previously obtained from a variety of marine environments. Archaeal clones exhibited higher similarities (83.25-100%) to sequences from other environments in the public database than did those (75.22-98.46%) of previously reported bacterial clones obtained from tidal flat sediment. The results of our study suggest that the archaeal community in tidal flat sediment is remarkably diverse.

Optimization of bacteriocin ST311LD production by Enterococcus faecium ST311LD, isolated from spoiled black olives.

Abstract: Bacteriocin ST311LD is approximately 2.3 kDa in size. Low levels of bacteriocin activity were recorded in BHI and M17 broth (800 AU/ml) and in 10% (w/v) soy milk (3,200 AU/ml). No bacteriocin production was recorded in 10% (w/v) molasses, despite good growth. Optimal levels (12,800 AU/ml) were detected in MRS broth which had been supplemented with tryptone (20.0 g/l), saccharose (5.0 or 10.0 g/l) or vitamin C (1 ppm). Increased potassium levels did not result in higher levels of activity, and glycerol (1.0 g/l) inhibited the production of bacteriocin ST311LD.

Isolation and characterization of bacteria associated with two sand dune plant species, Calystegia soldanella and Elymus mollis.

Abstract: Little is known about the bacterial communities associated with the plants inhabiting sand dune ecosystems. In this study, the bacterial populations associated with two major sand dune plant species, Calystegia soldanella (beach morning glory) and Elymus mollis (wild rye), growing along the costal areas in Tae-An, Chungnam Province, were analyzed using a culture-dependent approach. A total of 212 bacteria were isolated from the root and rhizosphere samples of the two plants, and subjected to further analysis. Based on the analysis of the 16S rDNA sequences, all the bacterial isolates were classified into six major phyla of the domain Bacteria. Significant differences were observed between the two plant species, and also between the rhizospheric and root endophytic communities. The isolates from the rhizosphere of the two plant species were assigned to 27 different established genera, and the root endophytic bacteria were assigned to 21. Members of the phylum Gammaproteobacteria, notably the Pseudomonas species, comprised the majority of both the rhizospheric and endophytic bacteria, followed by members of Bacteroidetes and Firmicutes in the rhizosphere and Alphaproteobacteria and Bacteroidetes in the root. A number of isolates were recognized as potentially novel bacterial taxa. Fifteen out of 27 bacterial genera were commonly found in the rhizosphere of both plants, which was comparable to 3 out of 21 common genera in the root, implying the host specificity for endophytic populations. This study of the diversity of culturable rhizospheric and endophytic bacteria has provided the basis for further investigation aimed at the selection of microbes for the facilitation of plant growth.

Molecular Characterization of TEM-type β-Lactamases Identified in Cold-Seep Sediments of Edison Seamount (South of Lihir Island, Papua New Guinea)

Abstract: To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.

The diversity of culturable organotrophic bacteria from local solar salterns.

Abstract: We isolated and cultured bacteria inhabiting solar saltern ponds in Taean-Gun, Chungnam Province, Korea. All of the isolated 64 strains were found to be moderately halophilic bacteria, growing in a salt range of 2-20 %, with an optimal concentration of 5% salt. Bacterial diversity among the isolated halophiles was evaluated via RFLP analyses of PCR-amplified 16S rDNAs, followed by phylogenetic analysis of the partial 16S rDNA sequences. The combination of restriction enzyme digestions with HaeIII, CfoI, MspI and RsaI generated 54 distinct patterns. A neighbor-joining tree of the partial 16S rDNA sequences resulted in the division of the 64 strains into 2 major groups, 45 strains of gamma-Proteobacteria (70.3%) and 19 strains of Firmicutes (29.7%). The alpha-Proteobacteria and Cytophaga-Flavobacterium-Bacterioides groups, which were repeatedly found to exist in thalassohaline environments, were not represented in our isolates. The gamma-Proteobacteria group consisted of several subgroups of the Vibrionaceae (37.5%), Pseudoalteromonadaceae (10.9%), Halomonadaceae (7.8%), Alteromonadaceae (7.8%), and Idiomarinaceae (6.3%). Members of Salinivibrio costicola (29.7%) were the most predominant species among all of the isolates, followed by Halobacillus treperi (12.5%). Additionally, three new species candidates were found, based on similarities of the 16S rDNA sequences to those of previously published species.

Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors: influence of culture conditions.

Abstract: The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, Cu2+ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.

Enhancing the intrinsic bioremediation of PAH-contaminated anoxic estuarine sediments with biostimulating agents.

Abstract: Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 microg/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 microg PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.

Degradation of crystalline cellulose by the brown-rot basidiomycete Fomitopsis palustris.

Abstract: This study demonstrated that the brown rot basidiomycete Fomitopsis palustris was able to degrade crystalline cellulose (Avicel). This fungus could also produce the three major cellulases (exoglucanases, endoglucanases, and beta-glucosidase) when the cells were grown on 2.0% Avicel. Avicel degraded by F. palustris showed a decrease in relative crystallinity from 83% to 78.5% after 14 days of incubation. The characterization study indicated that optimum pH was 4.5 and optimum temperature was 70 degrees C for exoglucanase (cellobiohydrolase) activity. Hydrolysis of Avicel by the crude enzyme from F. palustris yielded 1.6 mg/ml of glucose after 43 h, which corresponded to a cellulose conversion degree of 3.2%. Therefore, this study revealed for the first time that the brown rot basidiomycete F. palustris produces cellulases capable of yielding soluble sugars from crystalline cellulose.

Purification and characterization of manganese peroxidase of the white-rot fungus Irpex lacteus.

Abstract: The production of manganese peroxidase (MnP) by Irpex lacteus, purified to electrophoretic homogeneity by acetone precipitation, HiPrep Q and HiPrep Sephacryl S-200 chromatography, was shown to correlate with the decolorization of textile industry wastewater. The MnP was purified 11.0-fold, with an overall yield of 24.3%. The molecular mass of the native enzyme, as determined by gel filtration chromatography, was about 53 kDa. The enzyme was shown to have a molecular mass of 53.2 and 38.3 kDa on SDS-PAGE and MALDI-TOF mass spectrometry, respectively, and an isoelectric point of about 3.7. The enzyme was optimally active at pH 6.0 and between 30 and 40 degrees C. The enzyme efficiently catalyzed the decolorization of various artificial dyes and oxidized Mn (II) to Mn (III) in the presence of H(2)O(2). The absorption spectrum of the enzyme exhibited maxima at 407, 500, and 640 nm. The amino acid sequence of the three tryptic peptides was analyzed by ESI Q-TOF MS/MS spectrometry, and showed low similarity to those of the extracellular peroxidases of other white-rot basidiomycetes.

Nematicidal Activity and Chemical Component of Poria cocos

Abstract: Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.

Investigations on bacteria as a potential biological control agent of summer chafer, Amphimallon solstitiale L. (Coleoptera: Scarabaeidae).

Abstract: Studying the bacteria of hazardous insects allows the opportunity to find potentially better biological control agents. Therefore, in this study, bacteria from summer chafer (Amphimallon solstitiale L., Coleoptera: Scarabaeidae) we isolated and identified the insecticidal effects of bacteria isolated from A. solstitiale and Melolontha melolontha L. (common cockchafer, Coleoptera: Scarabaeidae) and the mixtures of these bacterial isolates were investigated on A. solstitiale larvae. Crystals from Bacillus sp. isolated from M. melolontha were also purified, and tested against the second and third-stage larvae of A. solstitiale. The bacterial isolates of A. solstitiale were identified as Pseudomonas sp., Pseudomonas sp., Bacillus cereus and Micrococcus luteus, based on their morphology, spore formation, nutritional features, and physiological and biochemical characteristics. The insecticidal effects of the bacterial isolates determined on the larvae of A. solstitiale were 90% with B. cereus isolated from A. solstitiale, and 75% with B. cereus, B. sphaericus and B. thuringiensis isolated from M. melolontha within ten days. The highest insecticidal effects of the mixed infections on the larvae of A. solstitiale were 100% both with B. cereus+B. sphaericus and with B. cereus+B. thuringiensis. In the crystal protein bioassays, the highest insecticidal effect was 65% with crystals of B. thuringiensis and B. sphaericus isolated from M. melolontha within seven days. Finally, our results showed that the mixed infections could be utilized as microbial control agents, as they have a 100% insecticidal effect on the larvae of A. solstitiale.

Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron.

Abstract: Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.

Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken.

Abstract: In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.