Inactivation of the lpcC gene alters surface-related properties and symbiotic capability of Bradyrhizobium japonicum.

UNLABELLED: We investigated the role of the Bradyrhizobium japonicum lpcC gene, encoding a mannosyl transferase, involved in the lipopolysaccharide (LPS) biosynthesis. The inactivation of the lpcC gene considerably altered the LPS structure and the cell surface properties. LPS analysis showed that the lpcC mutant JS715 had an abnormal LPS structure deficient in O-antigen. The cell surface hydrophobicity increased approximately threefold in JS715 compared to the wild type. The increased cell surface hydrophobicity is likely to be related with cell aggregation in the mutant culture. For the growth comparison, JS715 showed slower growth rate than the wild type. The motility of JS715 decreased in soft agar plates, but it showed enhanced biofilm-forming ability. Interestingly, JS715 was not able to nodulate the host legume soybean (Glycine max). This study shows not only that lpcC is involved in the biosynthesis of O-antigen in the B. japonicum LPS, but also that inactivation of the lpcC gene affects symbiotic capability of B. japonicum and surface-related properties such as cell hydrophobicity, biofilm formation and motility. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the role of the B. japonicum lpcC in nodulation with soybean and importance of cell surface hydrophobicity. The results also highlight that intact LPS is required for successful symbiosis between B. japonicum and soybeans. Our findings not only support previous studies emphasizing the necessity of LPS on the interaction between the two symbiotic partners, but also contribute to a better understanding of the symbiotic mechanisms.

Lack of O-polysaccharide enhances biofilm formation by Bradyrhizobium japonicum.

AIMS: To reveal the effects of the O-polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. METHODS AND RESULTS: Wild type and O-antigen-deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi-solid (0.3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3.5-fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. CONCLUSIONS: This study shows enhanced biofilm formation and decreased motility by the O-antigen-deficient mutant, suggesting that the lack of the O-polysaccharide of the rhizobial LPS is associated with biofilm-forming ability and movement. SIGNIFICANCE AND IMPACT OF THE STUDY: LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O-antigen-deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum.

Species diversity and relative abundance of vaginal lactic acid bacteria from women in Uganda and Korea.

AIMS: Lactobacilli play an important role in maintaining vaginal health of women. The aim of this study was to compare the species richness and relative abundance of Lactobacillus and other lactic acid bacteria in women of two geographically distant countries, Uganda and Korea. METHODS AND RESULTS: Vaginal samples were obtained from two women populations in Uganda and Korea. The Lactobacillus Rogosa SL agar was used for initial isolation of lactic acid bacteria. After phenotypic analyses, the 16S rRNA gene was amplified by polymerase-chain reaction and analysed by the BLAST program and phylogenetic tree construction. A total of 338 (128 Korean and 210 Ugandan) vaginal lactic acid bacterial strains were isolated, including five genera: Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Weissella. While Lactobacillus crispatus was common in both populations, Lactobacillus fermentum was common only in Korean women, and Lactobacillus gasseri, Lactobacillus reuteri and Lactobacillus vaginalis only in Ugandan women. Among other lactic acid bacteria, Weissella was more common in Ugandan, and Pediococcus in Korean women. All Weissella strains produced hydrogen peroxide, and all Pediococcus strains inhibited Candida species. CONCLUSION: Although many lactic acid bacteria colonize women, their species distributions may be different in women of geographically separated communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of species richness and relative abundance of vaginal lactic acid bacteria, including Lactobacillus, Pediococcus and Weissella, may lead to the design of better probiotic products as bacterial replacement therapy.

Carbamazepine-induced hypersensitivity syndrome occurring in a photodistributed pattern.

A 76- year- old man was commenced on carbamazepine for partial seizures. This was followed by the development of a rash in an apparently photodistributed pattern, fever, lymphadenopathy, eosinophilia, abnormal liver function tests and atypical lymphocytosis fulfilling the criteria for drug-induced hypersensitivity syndrome. Discontinuation of carbamazepine and application of topical steroid resulted in clearance of the rash, normalization of liver function tests and improvement in eosinophilia. The photodistributed pattern in this case of carbamazepine-induced hypersensitivity syndrome is of interest.

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences.

AIMS: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment. METHODS AND RESULTS: Thirty-five Lactobacillus strains from women from seven countries were isolated, and sequences from 16S rRNA genes were determined and compared with existing data in GenBank. A phylogenetic tree was achieved using the Neighbour-Joining method based on the analysis of 1465 nucleotides. The results showed that most vaginal isolates were L. crispatus, L. jensenii and L. gasseri. Some were L. vaginalis, L. fermentum, L. mucosae, L. paracasei and L. rhamnosus. Two isolates from a native American woman displayed distinct branches, indicating novel phylotypes. Few vaginal isolates matched food or environmental Lactobacillus species. CONCLUSIONS: Most women worldwide were colonized by three common Lactobacillus species: L. crispatus, L. jensenii and L. gasseri. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of vaginal Lactobacillus species richness and distribution in women worldwide may lead to the design of better probiotic products as bacterial replacement therapy.

Thin layer chromatographic determination of organic acids for rapid identification of bifidobacteria at genus level.

This study presents a simple and fast method for the identification of bifidobacteria using a thin layer chromatographic (TLC) analysis of the short chain fatty acids in a culture broth. When the chromatogram was sprayed with the indicator solution (methyl red-bromophenol blue in 70% ethanol), lactic acid exhibited two red spots, and acetic acid, propionic acid, and butyric acid all produced blue spots. Succinic acid and citric acid produced yellow and dark yellow spot, respectively. In addition, these organic acids showed different R(f) values. The total time taken to analyze the organic acids in the 10 bacterial culture broths using the proposed method was approximately 50 min. The proposed TLC method was used to analyze the organic acids in culture broths of the following strains, five Bifidobacterium species. (Bifidobacterium longum, B. breve, B. infantis, B. bifidum, and B. adolescentis) and five other lactic acid bacteria strains (Lactobacillus casei, L. bulgaricus, L. acidophilus, Streptococcus thermophilus, and S. lactis). Both spots of lactic acid and acetic acid were detected on all the TLC plates from the five bifidobacterial culture broths. The five other lactic acid bacterial culture broths, however, only exhibited lactic acid spots. Accordingly, the proposed TLC method would appear to be a useful tool for rapid identification of Bifidobacterium spp. at the genus level.

Simultaneous detection of gfp-marked Moraxella sp. G21r and lux-marked Ralstonia eutrophas H850Lr using most-probable-number method.

The green fluorescent protein encoded by gfp gene and the luminescent protein encoded by luxAB genes were used as markers to detect p-nitrophenol (PNP)-degrading Moraxella sp. G21r and polychlorinated biphenyl (PCB)-degrading Ralstonia eutrophas H850Lr cells, respectively, in mixed liquid cultures and in soil samples using a most-probable-number (MPN) assay. Population estimates for both gfp-marked G21r and lux-marked H850Lr by using MPN assays were similar to viable colony counts. The MPN assay with microtiter plates permitted the simultaneous detection of fluorescent and luminescent bacteria in soil samples faster than conventional plate counting.

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene.

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.

Altered cell surface hydrophobicity of lipopolysaccharide-deficient mutant of Bradyrhizobium japonicum.

The authors previously isolated a lipopolysaccharide (LPS) deficient Tn5-mutant of Bradyrhizobium japonicum, and subsequently isolated the LPS gene region. In this study the LPS deficiency of B. japonicum was studied in terms of its cell surface characteristics. By monitoring the kinetics of the partition with hexadecane the LPS-mutant was found to be far more hydrophobic than the wild type strain; the partition coefficients were 3.19 min(-1) for the mutant, as compared with only 1.40 min(-1) for the wild type. When the mutant was transformed with the cloned LPS gene, the transformant regained the wild type phenotypes, including the cell surface hydrophobicity (CSH) and LPS profile. A polyacrylamide gel electrophoretic analysis of LPS demonstrated that the O-antigenic part of LPS was completely absent in the mutant. The LPS-mutant of B. japonicum was visually distinguishable from the wild type after a simple centrifugation of the cells.

Using a green fluorescent protein gene-labeled p-nitrophenol-degrading Moraxella strain to examine the protective effect of alginate encapsulation against protozoan grazing.

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.

The effect of extracellular cyclic AMP on differentiation inducing factor (DIF)-dependent prestalk cell gene expression in monolayers of Dictyostelium is complex.

An earlier finding that the ecmA and ecmB prestalk cell specific genes exhibited very different responses to cyclic AMP prompted the suggestion that cyclic AMP might act as the major spatial regulator of the prestalk cell developmental pattern in Dictyostelium. A more detailed kinetic analysis in monolayers of Dictyostelium has revealed that cyclic AMP inhibits the rate of expression of all three differentiation inducing factor (DIF) inducible genes, ecmA, ecmB and pDd26. After prolonged incubation, however, cyclic AMP enhances the levels of both ecmA and ecmB mRNAs, and nuclear run-on experiments suggest that cyclic AMP inhibits the degradation of both mRNA species. This complex response to cyclic AMP can explain the differential effects reported previously. Thus depending upon the experimental conditions, cyclic AMP can either enhance or reduce the apparent steady state level of a specific mRNA species. These results are not compatible with the earlier proposal that cyclic AMP is a spatial regulator of the prestalk developmental pattern. Although ecmA and ecmB accumulate rapidly in response to DIF, there is a lag in the accumulation of pD26 mRNA and the induction requires protein synthesis. These results suggest that pDd26 transcription requires the accumulation of an additional factor(s). Inhibition of pDd26 mRNA accumulation by cyclic AMP also occurs during the lag period, suggesting the possibility that cyclic AMP inhibits the accumulation of the, as yet, unknown factor. The inhibitory effect of cyclic AMP on pDd26 gene expression is unaffected by caffeine, suggesting that inhibition does not involve adenylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of a gene region from Bradyrhizobium japonicum essential for lipopolysaccharide synthesis.

The gene region cloned from a lipopolysaccharide (LPS) mutant carrying the Tn5 and flanking DNA sequences was used as a probe to screen a gene bank prepared from wild-type Bradyrhizobium japonicum strain 61A101C and to isolate the corresponding wild-type LPS-gene region. By cross-hybridization experiments the LPS-gene region did not appear to be closely linked to previously cloned nodulation genes. A detailed restriction map of the LPS-gene region (5.5-kb EcoRI genomic fragment) was established and the mutation site was localized to be in a 300-bp PvuI/PstI restriction fragment. In genomic Southern-blot analysis of various rhizobia, the LPS-gene region was found to be conserved among all the slow-growing bradyrhizobia, but not the fast-growing rhizobia. The different groups of slow-growing bradyrhizobia are polymorphic for restriction-fragment length at the LPS-gene region.

Binding of an endosperm-specific nuclear protein to a maize beta-zein gene correlates with zein transcriptional activity.

Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was found only in nuclei from developing endosperm tissue. Mixing of nuclear extracts demonstrated that seedling tissue contained undetectable amounts of beta-1, rather than having an inhibitor for formation of the beta-1/DNA complex. Chemical footprinting analysis localized the beta-1 recognition site to a 22 bp sequence flanked by CCAT and TATA boxes. The apparent molecular mass of beta-1 was determined to be 29 kDa by southwestern blotting. Based on in vitro binding assays, the greatest concentration of the beta-1 in endosperm nuclei is at 16 days after pollination, which coincides with the time of highest transcriptional activity of the beta-zein gene. These results suggest that beta-1 may act as a tissue-specific, trans-acting regulator of the expression of the beta-zein gene in developing maize endosperm.

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation.

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three non-auxotrophic Nod- mutants and one His-Nod- mutant of USDA110. Homogenotization of the cloned fragments into wild-type strain USDA110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.