Identification of Free-Living Amoebas in Tap Water of Buildings with Storage Tanks in Korea.

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

Chromosome I controls chromosome II replication in Vibrio cholerae.

Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication.

Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II.

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.

Chromosome dynamics in multichromosome bacteria.

On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space.

Participation of chromosome segregation protein ParAI of Vibrio cholerae in chromosome replication.

Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.

Enhanced display of lipase on the Escherichia coli cell surface, based on transcriptome analysis.

A cell surface display system was developed using Escherichia coli OmpC as an anchoring motif. The fused Pseudomonas fluorescens SIK W1 lipase was successfully displayed on the surface of E. coli cells, and the lipase activity could be enhanced by the coexpression of the gadBC genes identified by transcriptome analysis.

Transcriptome and proteome analyses of adaptive responses to methyl methanesulfonate in Escherichia coli K-12 and ada mutant strains.

BACKGROUND: The Ada-dependent adaptive response system in Escherichia coli is important for increasing resistance to alkylation damage. However, the global transcriptional and translational changes during this response have not been reported. Here we present time-dependent global gene and protein expression profiles following treatment with methyl methanesulfonate (MMS) in E. coli W3110 and its ada mutant strains. RESULTS: Transcriptome profiling showed that 1138 and 2177 genes were differentially expressed in response to MMS treatment in the wild-type and mutant strains, respectively. A total of 81 protein spots representing 76 nonredundant proteins differentially expressed were identified using 2-DE and LC-MS/MS. In the wild-type strain, many genes were differentially expressed upon long-exposure to MMS, due to both adaptive responses and stationary phase responses. In the ada mutant strain, the genes involved in DNA replication, recombination, modification and repair were up-regulated 0.5 h after MMS treatment, indicating its connection to the SOS and other DNA repair systems. Interestingly, expression of the genes involved in flagellar biosynthesis, chemotaxis, and two-component regulatory systems related to drug or antibiotic resistance, was found to be controlled by Ada. CONCLUSION: These results show in detail the regulatory components and pathways controlling adaptive response and how the related genes including the Ada regulon are expressed with this response.

Transcript and protein level analyses of the interactions among PhoB, PhoR, PhoU and CreC in response to phosphate starvation in Escherichia coli.

The responses to phosphate starvation by PhoR-PhoB two-component regulatory system are also affected by other regulatory systems in Escherichia coli. The interactions among PhoB, PhoR, PhoU and CreC were investigated at transcript and protein levels using real-time PCR and fluorescence resonance energy transfer analyses. CreC showed an interaction with PhoB, before an interaction between PhoB and PhoR, suggesting that this acts as a constant sensor for the early response to phosphate starvation. PhoU was found to interact with neither PhoB nor PhoR, while this acted as a phosphate starvation-inducible protein. These results showed the detailed time- and phosphate concentration-dependent interactions among PhoB, PhoR, PhoU and CreC in response to phosphate starvation in E. coli.

Transcriptome analysis of phosphate starvation response in Escherichia coli.

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.

Novel gene members in the Pho regulon of Escherichia coli.

The transcriptome profiles of the wild-type and the phoB mutant strains were compared at the time point showing the highest expression levels of the phoB and phoR genes under a P-limiting condition. Among the 18 new putative genes that were found to be under the control of the PhoB transcriptional regulator, five genes that contain the consensus Pho box were identified by sequence analysis. A reporter gene assay was carried out by fusing the upstream regions of these genes to the promoterless enhanced green fluorescent protein gene, followed by expression. It was found that the expressions of the amn (AMP nucleosidase), yibD (metal ion stress response gene) and ytfK (hypothetical protein) genes were activated by PhoB. These results indicate the additional roles of PhoB as a global regulator.

Chromosome segregation proteins of Vibrio cholerae as transcription regulators.

ABSTRACT Bacterial ParA and ParB proteins are best known for their contribution to plasmid and chromosome segregation, but they may also contribute to other cell functions. In segregation, ParA interacts with ParB, which binds to parS centromere-analogous sites. In transcription, plasmid Par proteins can serve as repressors by specifically binding to their own promoters and, additionally, in the case of ParB, by spreading from a parS site to nearby promoters. Here, we have asked whether chromosomal Par proteins can likewise control transcription. Analysis of genome-wide ParB1 binding in Vibrio cholerae revealed preferential binding to the three known parS1 sites and limited spreading of ParB1 beyond the parS1 sites. Comparison of wild-type transcriptomes with those of ΔparA1, ΔparB1, and ΔparAB1 mutants revealed that two out of 20 genes (VC0067 and VC0069) covered by ParB1 spreading are repressed by both ParB1 and ParA1. A third gene (VC0076) at the outskirts of the spreading area and a few genes further away were also repressed, particularly the gene for an outer membrane protein, ompU (VC0633). Since ParA1 or ParB1 binding was not evident near VC0076 and ompU genes, the repression may require participation of additional factors. Indeed, both ParA1 and ParB1 proteins were found to interact with several V. cholerae proteins in bacterial and yeast two-hybrid screens. These studies demonstrate that chromosomal Par proteins can repress genes unlinked to parS and can do so without direct binding to the cognate promoter DNA. IMPORTANCE Directed segregation of chromosomes is essential for their maintenance in dividing cells. Many bacteria have genes (par) that were thought to be dedicated to segregation based on analogy to their roles in plasmid maintenance. It is becoming clear that chromosomal par genes are pleiotropic and that they contribute to diverse processes such as DNA replication, cell division, cell growth, and motility. One way to explain the pleiotropy is to suggest that Par proteins serve as or control other transcription factors. We tested this model by determining how Par proteins affect genome-wide transcription activity. We found that genes implicated in drug resistance, stress response, and pathogenesis were repressed by Par. Unexpectedly, the repression did not involve direct Par binding to cognate promoter DNA, indicating that the repression may involve Par interactions with other regulators. This pleiotropy highlights the degree of integration of chromosomal Par proteins into cellular control circuitries.

Association between white matter hyperintensity severity and cognitive impairment according to the presence of the apolipoprotein E (APOE) ε4 allele in the elderly: retrospective analysis of data from the CREDOS study.

OBJECTIVE: To investigate the effect of white matter hyperintensity (WMH) severity on cognitive function according to presence of the apolipoprotein E (APOE) ε4 allele. METHOD: From participants in a nationwide, multicenter, hospital-based cohort study of dementia by the Clinical Research Center for Dementia of South Korea (November 2005 to December 2011), data for 5,077 elderly subjects (mean [SD] age = 71.37 [8.40] years) who had available data for APOE genotype and WMH severity were studied retrospectively. We used the diagnostic criteria for mild cognitive impairment proposed by Petersen et al; the diagnostic criteria for vascular dementia included in DSM-IV; and, for probable Alzheimer's disease, the criteria issued by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association. WMH severity was evaluated using modified criteria of Fazekas et al and Scheltens et al using T2 axial or fluid-attenuated inversion recovery magnetic resonance images, yielding 3 groups for WMH severity level. APOE genotype was determined by analysis of venous blood, and all participants were classified into 2 groups depending on presence or absence of the APOE ε4 allele. The Seoul Neuropsychological Screening Battery-Dementia Version was used for all subjects. Cognitive impairment, classified by 6 cognitive test scores, was the primary outcome measure. Using multiple logistic regression, we investigated which cognitive domains were associated with WMH severity and the APOE ε4 allele, and, using analysis of covariance, we examined the interaction effects of these 2 factors on cognitive test scores. RESULTS: After multivariable adjustments, logistic regression analyses showed that WMH severity was associated with higher odds of cognitive impairment on frontal/executive function tests in both APOE ε4 carriers (odds ratio [OR] = 2.49; 95% CI, 1.65-3.76) and noncarriers (OR = 2.36; 95% CI, 1.83-3.03). WMH severity was not significantly associated with memory function in APOE ε4 carriers: for verbal memory, ε4 noncarriers had an OR of 1.44 (95% CI, 1.13-1.84), and ε4 carriers had an OR of 1.36 (95% CI, 0.87-2.04); for visuospatial memory, ε4 noncarriers had an OR of 1.86 (95% CI, 1.45-2.37), and ε4 carriers had an OR of 1.35 (95% CI, 0.89-2.04). Moreover, a significant interaction effect between APOE ε4 and WMH severity was confirmed on memory tests by analysis of covariance (verbal memory: F = 3.40, P = .033; visuospatial memory: F = 8.49, P < .001). CONCLUSIONS: Severe WMHs appear to be predominantly associated with frontal/executive dysfunction, irrespective of APOE ε4 allele presence. WMH severity and APOE ε4 had an interactive effect on memory function, with WMH severity affecting memory impairment only in APOE ε4 noncarriers.

Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor.

A polyphasic study was carried out to determine the taxonomic position of two aerobic, cyanide-degrading bacterial strains, designated L61T and L22, which had been isolated from a bioreactor for the treatment of nickel-complexed cyanide. The two isolates exhibited almost identical taxonomic characteristics. Phylogenetic analysis inferred from comparative 16S rRNA gene sequences indicated that the isolates fall in a sublineage of the genus Rhizobium comprising the type strains of Rhizobium giardinii, Rhizobium radiobacter, Rhizobium rubi, Rhizobium larrymoorei, Rhizobium vitis, Rhizobium undicola, Rhizobium loessense, Rhizobium galegae and Rhizobium huautlense. Cells of the two isolates are Gram-negative, aerobic, motile and non-spore-forming rods (0.6-0.7x1.1-1.3 microm), with peritrichous flagella. The DNA G+C content is 60.1-60.9 mol%. Cellular fatty acids are C(16 : 0) (2.2-3.3 %), C(18 : 0) (2.1-3.2 %), C(19 : 0) cyclo omega8c (9.9-16.8 %), C(20 : 3)omega6,9,12c (2.7-3.3 %), summed feature 3 (7.2-7.7 %) and summed feature 7 (67.8-73.7 %). The strains formed nodules on a legume plant, Medicago sativa. A nifH gene encoding denitrogenase reductase, the key component of the nitrogenase enzyme complex, was detected in L61T by PCR amplification by using a nifH-specific primer system. Strains L61T and L22 were distinguished from the type strains of recognized Rhizobium species in the same sublineage based on low DNA-DNA hybridization values (2-4 %) and/or a 16S rRNA gene sequence similarity value of less than 96 %. Moreover, some phenotypic properties with respect to substrate utilization as a carbon or nitrogen source, antibiotic resistance and growth conditions could be used to discriminate L61T and L22 from Rhizobium species in the same sublineage. Based on the results obtained in this study, L61T and L22 are considered to be representatives of a novel species of Rhizobium, for which the name Rhizobium daejeonense sp. nov. is proposed. The type strain is L61T (=KCTC 12121T=IAM 15042T=CCBAU 10050T).