Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans.

We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.

Whole-genome expression profiling of Bradyrhizobium japonicum in response to hydrogen peroxide.

Bradyrhizobium japonicum, a nitrogen-fixing bacterium in soil, establishes a symbiotic relationship with the leguminous soybean plant. Despite a mutualistic association between the two partners, the host plant produces an oxidative burst to protect itself from the invasion of rhizobial cells. We investigated the effects of H(2)O(2)-mediated oxidative stress on B. japonicum gene expression in both prolonged exposure (PE) and fulminant shock (FS) conditions. In total, 439 and 650 genes were differentially expressed for the PE and FS conditions, respectively, at a twofold cut-off with q < 0.05. A number of genes within the transport and binding proteins category were upregulated during PE and a majority of those genes are involved in ABC transporter systems. Many genes encoding ? factors, global stress response proteins, the FixK(2) transcription factor, and its regulatory targets were found to be upregulated in the FS condition. Surprisingly, catalase and peroxidase genes which are typically expressed in other bacteria under oxidative stress were not differentially expressed in either condition. The isocitrate lyase gene (aceA) was induced by fulminant H(2)O(2) shock, as was evident at both the transcriptional and translational levels. Interestingly, there was no significant effect of H(2)O(2) on exopolysaccharide production at the given experimental conditions.

Genome-wide transcriptional and physiological responses of Bradyrhizobium japonicum to paraquat-mediated oxidative stress.

The rhizobial bacterium Bradyrhizobium japonicum functions as a nitrogen-fixing symbiont of the soybean plant (Glycine max). Plants are capable of producing an oxidative burst, a rapid proliferation of reactive oxygen species (ROS), as a defense mechanism against pathogenic and symbiotic bacteria. Therefore, B. japonicum must be able to resist such a defense mechanism to initiate nodulation. In this study, paraquat, a known superoxide radical-inducing agent, was used to investigate this response. Genome-wide transcriptional profiles were created for both prolonged exposure (PE) and fulminant shock (FS) conditions. These profiles revealed that 190 and 86 genes were up- and downregulated for the former condition, and that 299 and 105 genes were up- and downregulated for the latter condition, respectively (>2.0-fold; P < 0.05). Many genes within putative operons for F(0)F(1)-ATP synthase, chemotaxis, transport, and ribosomal proteins were upregulated during PE. The transcriptional profile for the FS condition strangely resembled that of a bacteroid condition, including the FixK(2) transcription factor and most of its response elements. However, genes encoding canonical ROS scavenging enzymes, such as superoxide dismutase and catalase, were not detected, suggesting constitutive expression of those genes by endogenous ROS. Various physiological tests, including exopolysaccharide (EPS), cellular protein, and motility characterization, were performed to corroborate the gene expression data. The results suggest that B. japonicum responds to tolerable oxidative stress during PE through enhanced motility, increased translational activity, and EPS production, in addition to the expression of genes involved in global stress responses, such as chaperones and sigma factors.

Screening and selection of stress resistant Lactobacillus spp. isolated from the marine oyster (Crassostrea gigas).

We attempted to isolate Lactobacillus spp. from the marine oyster (Crassostrea gigas) and select stress resistant strains for development of a future marine aquaculture feed adjuvant. A total of 83 lactobacilli strains were isolated from oyster. They were all Gram-positive, rod-shaped and catalase-negative. By performing a stress resistance assay, we selected eighteen isolates. Based on 16S rRNA gene sequencing, Lactobacillus paracasei was the most prevalent species among the selected isolates. The in vitro antagonistic effect of the selected strains against fish pathogens was assayed by measurement of inhibition diameters. Except for MH44, MH51, MH53 and MH62, most of the isolates showed inhibition of Vibrio alginolyticus and Vibrio proteolyticus (diameters over 15 mm). Lactobacillus rhamnosus MH22 was selected as the most stress resistant strain showing the MICs of 1.8 M NaCl, 14% ethanol and 0.014% hydrogen peroxide. L. rhamnosus MH22 isolated from oyster has a potential to be applied as a microbial feed adjuvant for marine aquaculture.

Heavy metal and antibiotic co-resistance in Vibrio parahaemolyticus isolated from shellfish.

Vibrio parahaemolyticus is a major gastroenteritis-causing pathogen in Korea. Recent studies have reported that heavy metal and antimicrobial resistance in bacteria are related. In this study, we investigated heavy metal and antimicrobial resistance in wild strains of V. parahaemolyticus. First, we isolated and characterized 38 V. parahaemolyticus strains (toxR-positive) from shellfish collected from the West Sea of Korea between May and November 2018. Antibiotic and heavy metal resistance in the 38 strains were tested by disk diffusion assay and broth dilution assay, respectively. Then, we selected seven strains that showed resistance to cobalt (Co2+) and copper (Cu2+), to examine the relationship between heavy metal resistance and antimicrobial resistance. After heavy metal (Co2+ and Cu2+) pretreatment, the seven strains exhibited increased resistance to kanamycin, streptomycin, tetracycline, and gentamycin. Likewise, antimicrobial pretreatment resulted in increased heavy metal tolerance.

Heat Adaptation Improved Cell Viability of Probiotic Enterococcus faecium HL7 upon Various Environmental Stresses.

The production of viable functional probiotics presupposes stability of strain features in the final product. In previous studies, Enterococcus faecium HL7 was found to have relatively higher cell viability after freeze-drying and the long-lasting resistance to heat (60 °C) as well as higher antimicrobial activities against some of fish and human pathogens among isolated strains. For heat adaptation, E. faecium HL7 cells were exposed to 52 °C for 15 min. After adaption, slight decreases of unsaturated membrane fatty acid ratios were confirmed through fatty acid analysis. Upon subsequent exposure to various stress conditions such as H2O2 (0.01%), ethanol (20%), acid (pH 3), and alkali (pH 12), the survival rate of heat-adapted HL7 was 103-105-fold higher than that of non-adapted one. These results highlight the potential of preconditioning treatments for maximizing survival of probiotic bacteria during development of probiotic functional foods. The cross-protection afforded by acid against thermal stress may indicate that certain common protective mechanisms are induced by both heat and acid stress. These results can be applied to enhancing the cell viability during live cell formulation of E. faecium HL7 to be used as a potential probiotics in aquaculture.

Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean.

The galE gene from Bradyrhizobium japonicum 61A101C, a soybean endosymbiont, was cloned and characterized. Its deduced amino-acid sequence showed a high similarity with that of other rhizobia. Functional identification of the galE gene was achieved by complementation of a galE mutant strain, PL2, with a series of pKM subclones. Disruption of the B. japonicum galE gene affects the lipopolysaccharide profile compared with that of the wild type, suggesting that galE is responsible for alteration of lipopolysaccharide structure. Examination of nodule formation by the wild-type and galE mutant revealed that the former displayed normal nodule development on soybean roots, whereas the latter showed no nodule formation at all time points examined except for 20 days after inoculation when <10% of soybean formed pseudo-nodules.

Optimization of electrotransformation conditions for Propionibacterium acnes.

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.

Possible Probiotic Lactic Acid Bacteria Isolated from Oysters (Crassostrea gigas).

We attempted to isolate lactic acid bacteria (LAB) from the marine oyster (Crassostrea gigas) and selected several environmental stress-resistant isolates for the development of a future probiotic adjuvant for marine aquaculture. Twenty-six presumptive LAB isolates were extracted from oysters and screened (by an agar diffusion assay) for antimicrobial activity toward various pathogens: Vibrio parahaemolyticus, Streptococcus iniae, and Edwardsiella tarda. Eight isolates had an antibacterial activity toward V. parahaemolyticus; in particular, 6 isolates showed a growth-inhibitory activity, with inhibition zone diameters > 15 mm. Of these, 5 isolates (JL17, JL18, JL28, HL7, and HL32) were also active against S. iniae and E. tarda. Enterococcus faecium HL7 was selected as the isolate most resistant to environmental stressors: the minimum NaCl, ethanol, and hydrogen peroxide concentrations at which HL7 cells lost their viability were 1.9 M, 11%, and 0.013%, respectively. When an antibiotic sensitivity test was performed on E. faecium HL7, this isolate was found to be resistant to trimethoprim/sulfamethoxazole, cephalothin, ampicillin, rifampin, gentamicin, cefotaxime, cefepime, cefotetan, nalidixic acid, and kanamycin. While the oyster model studies provided indication that E. faecium HL7 could be a good candidate as biocontrol agent against V. vulnificus, further optimization is needed in the actual animal rearing situation.

Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from oysters in Korea.

Vibrio parahaemolyticus, found frequently in oysters and other seafoods, is the most prevalent gastroenteritis-causing pathogen in Korea and other Asian countries. It is associated exclusively with the consumption of raw or improperly cooked contaminated seafood, especially oysters. In this study, we isolated and characterized 59 V. parahaemolyticus strains (toxR-positive) from May to October 2016 in shellfish-harvesting areas off the west coast of Korea. The results revealed that none of the isolates contained the tdh and trh toxicity genes. The multiple antibiotic resistance (MAR) value of most isolates was 0.32, but it was as high as 0.69 in one isolate strain. Moreover, when resistance to heavy metals was examined, the majority of the isolates displayed resistance to Ba2+ (98.3%), Co3+ (28.8%), Cd2+ (16.9%), and Cu2+ (13.6%). Interestingly our data revealed that tolerance to heavy metals was prevalent in the V. parahaemolyticus strains with more than two antibiotic resistance phenotypes.

In Vitro Probiotic Properties of Lactobacillus salivarius MG242 Isolated from Human Vagina.

Vulvovaginal candidiasis (VVC) is a very common infection worldwide that is mainly caused by Candida albicans. In a previous study, we showed that Lactobacillus salivarius MG242 has anti-Gardnerella vaginalis activity. In this study, we investigated the potential of using L. salivarius MG242 for biocontrol of C. albicans. In line with the results from a spot overlay assay, MG242 inhibited the growth of C. albicans by 99.99 ± 0.01% in co-culture, suggesting that L. salivarius MG242 has the potential to be developed into a probiotic formula to treat or prevent VVC. Accelerated storage tests using dehydrated live cell powder at 50, 60, and 70 °C were performed, and the results showed that immobilization with 10% skim milk effectively increased the thermal resistance of entrapped microorganisms, resulting in sevenfold longer shelf-life than the control (in PBS). Lower storage temperatures also increased the shelf-life up to 8.31 months.

In vitro probiotic properties of vaginal Lactobacillus fermentum MG901 and Lactobacillus plantarum MG989 against Candida albicans.

Candida albicans is the most important Candida species causing vulvovaginal candidiasis (VVC). We investigated the potential of the probiotic strains Lactobacillus fermentum MG901 and L. plantarum MG989 towards control of C. albiacns. Cell viability tests following co-culturing with lactobacilli revealed that C. albicans cells lost metabolic activity and were eventually killed. Further studies revealed that MG901 and MG989 had high surface hydrophobicity that enhanced its adhesion ability to epithelial cell. The MG901 and MG989 showed coaggregation with E. coli and C. albicans to affect their adhesion and colonization. The adhesion of MG901 and MG989 to HT-29 cell and its inhibition of E. coli and C. albicans adherence to these cells were demonstrated. These incidences provided evidence of the possible colonization of MG901 and MG989 that would prevent binding and growth of E. coli and C. albicans onto intestinal epithelial cells. Following daily administration of 108 CFU of viable MG901 and MG989 orally, the animals' feces were examined for bacterial excretion. The potential probiotic MG901 and MG989 were found to persist for up to 6 days in the feces of mice. In conclusion, L. fermentum MG901 and L. plantarum MG989 have the potential to inhibit the yeast growth, which could possibly have played an important role in helping to clear VVC in vivo.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes.

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.

Biocementation of Concrete Pavements Using Microbially Induced Calcite Precipitation.

In this study, the feasibility of introducing calcite-forming bacteria into concrete pavements to improve their mechanical performance was investigated. Lysinibacillus sphaericus WJ-8, which was isolated in a previous study and is capable of exhibiting high urease activity and calcite production, was used. When analyzed via scanning electron microscopy (SEM) and X-ray diffraction, WJ-8 showed a significant amount of calcite precipitation. The compressive strength of cement mortar mixed with WJ-8 cells and nutrient medium (urea with calcium lactate) increased by 10% compared with that of the controls. Energy dispersive x-ray spectroscopy analyses confirmed that the increase in strength was due to the calcite formed by the WJ-8 cells.

Reductive dechlorination of polychlorinated biphenyls as affected by natural halogenated aromatic compounds.

We investigated the effects of halogenated aromatic compounds (HACs) including naturally occurring ones (L-thyroxine, 3-chloro-L-tyrosine, 5-chloroindole, 2-chlorophenol, 4-chlorophenol and chlorobenzene) on polychlorinated biphenyl (PCB) dechlorination in sediment cultures. A PCB-dechlorinating enrichment culture of sediment microorganisms from the St. Lawrence River was used as an initial inoculum. When the culture was inoculated into Aroclor 1248 sediments amended with each of the six HACs, the extent of dechlorination was not enhanced by amendment with HACs. The dechlorination patterns in the HAC-amended sediments were nearly identical to that of the HAC-free sediments except the 3-chloro-L-tyrosine-amended ones where no dechlorination activity was observed. When these sediment cultures were transferred into fresh sediments with the same HACs, the dechlorination specificities remained the same as those of the initial inoculations. Thus, in the present study, the substrate range of the highly selected enrichment culture could not be broadened by the HACs. It appears that HACs affect PCB dechlorination mainly through population selection rather than enzyme induction of single population.

Antibiotic and heavy metal resistance in Shewanella putrefaciens strains isolated from shellfishes collected from West Sea, Korea.

The aim of the present study was to determine the antibiotic resistance patterns and distribution of heavy-metal resistance in Shewanella putrefaciens strains isolated from shellfishes collected from West Sea; and to determine the relationship, if any, between antibiotic and heavy-metal resistance in these strains. Among the 15 strains isolated, two strains, SY1 and SY2, showed heavy-metal resistance in addition to high resistance to seven antibiotics: cephalothin, gentamicin, erythromycin, vancomycin, ampicillin, rifampicin, and streptomycin. We conclude that heavy-metal contamination imposes long-term, widespread, and recalcitrant selection pressure, which potentially contributes to the maintenance and spread of antibiotic resistance factors in bacteria. Moreover, this fact holds both environmental and clinical importance.

Formations of calcium carbonate minerals by bacteria and its multiple applications.

Biomineralization is a naturally occurring process in living organisms. In this review, we discuss microbially induced calcium carbonate precipitation (MICP) in detail. In the MICP process, urease plays a major role in urea hydrolysis by a wide variety of microorganisms capable of producing high levels of urease. We also elaborate on the different polymorphs and the role of calcium in the formation of calcite crystal structures using various calcium sources. Additionally, the environmental factors affecting the production of urease and carbonate precipitation are discussed. This MICP is a promising, eco-friendly alternative approach to conventional and current remediation technologies to solve environmental problems in multidisciplinary fields. Multiple applications of MICP such as removal of heavy metals and radionuclides, improve the quality of construction materials and sequestration of atmospheric CO2 are discussed. In addition, we discuss other applications such as removal of calcium ions, PCBs and use of filler in rubber and plastics and fluorescent particles in stationary ink and stationary markers. MICP technology has become an efficient aspect of multidisciplinary fields. This report not only highlights the major strengths of MICP, but also discusses the limitations to application of this technology on a commercial scale.

Antimicrobial susceptibility of Vibrio alginolyticus isolated from oyster in Korea.

Pathogenic Vibrio alginolyticus, a cause of severe infection in shellfish, as well as in humans, has been found at high frequency around all coastal areas of Korea. The aim of this study was to determine the occurrence of V. alginolyticus, to identify the strains isolated from oysters in West Sea, and to investigate their antimicrobial resistance profiles. Biochemical analyses of the 90 initially recovered presumptive V. alginolyticus colonies indicated that 16 isolates were V. alginolyticus. PCR analysis to detect the presence of the gyrB gene confirmed that 15 (93.8 %) of the 16 isolates were V. alginolyticus. These 15 isolates had the following profiles of resistance against 16 antibiotics: all isolates were resistant to ampicillin and vancomycin, and 26.7 % of the isolates exhibited resistance to cephalothin. A large number of isolates showed intermediate resistance to erythromycin (100 %) and rifampin (73.3 %). Five (33.3 %) of the V. alginolyticus isolates demonstrated multiple resistance to at least three antimicrobials.

Biosequestration of copper by bacteria isolated from an abandoned mine by using microbially induced calcite precipitation.

Abandoned mine sites are frequently polluted with high concentrations of heavy metals. In this study, 25 calcite-forming bacteria were newly isolated from the soil of an abandoned metal mine in Korea. Based on their urease activity, calcite production, and resistance to copper toxicity, four isolates were selected and further identified by 16S rRNA gene sequencing. Among the isolates, Sporosarcina soli B-22 was selected for subsequent copper biosequestration studies, using the sand impermeability test by production of calcite and extracellular polymeric substance. High removal rates (61.8%) of copper were obtained when the sand samples were analyzed using an inductively coupled plasma-optical emission spectrometer following 72 h of incubation. Scanning electron microscopy showed that the copper carbonate precipitates had a diameter of approximately 5-10 µm. X-ray diffraction further confirmed the presence of copper carbonate and calcium carbonate crystals.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from oysters in Korea.

Vibrio parahaemolyticus is the most prevalent gastroenteritis-causing pathogen in Korea and in some other Asian countries. It is frequently found in oysters and other seafood. This study monitored changes in the prevalence of V. parahaemolyticus and environmental parameters in oyster aquaculture environments in Korea. From June to October 2014, we tested oysters (Crassostrea gigas) from shellfish-harvesting areas off the west coast of Korea. These 71 isolates were the sum of 16 (22.5%), 19 (26.8%), 23 (32.4%), and 13 (18.3%) isolates collected in July, August, September, and October, respectively. These 71 isolates had the following profiles of resistance against 16 antibiotics: all isolates were resistant to ampicillin and vancomycin, and 52.2, 50.7, and 50.7% of isolates exhibited resistance to cephalothin, rifampin, and streptomycin, respectively. PCR analysis for the presence of the species-specific toxR gene confirmed that 38 (53.5%) of the total 71 isolated strains were positive for V. parahaemolyticus. In PCR analysis for virulence of V. parahaemolyticus, of the 71 isolates tested in the present study, only 38 (53.5%) were positive for the trh virulence gene and 71 (100%) was negative for the tdh virulence gene.