-
[show abstract]
[hide abstract]
ABSTRACT: In Salmonella enterica serovar Typhimurium, many of the genes required for intestinal penetration and invasion of host cells are encoded within the Salmonella pathogenicity island 1 (SPI1). The expression of invF, which is a positive transcriptional activator of SPI1, is controlled by HilA-dependent (invF-1) and HilC/D-dependent (invF-2) promoters. Transcriptional analysis of invF revealed that the invF-2 promoter (P(invF-2)) was not activated when cells were grown in standing culture conditions (which are known to induce SPI1) and that hilD mutation decreased the expression of P(invF-2) only in shaking culture conditions. In the absence of invF-1 promoter (P(invF-1)), P(invF-2) promoted InvF production and sipC expression (which is regulated by InvF) in shaking culture conditions. An analysis of the transcription patterns of plasmids harboring the lacZY reporter gene under various P(invF-2) derivatives with truncations or mutations revealed that the downstream region of the P(invF-2) transcription start site (i.e., +148 to +363) plays a role in repressing P(invF-2) in standing culture and in HilD-dependent activation of P(invF-2) in shaking culture conditions. The expression of invH overlaps with P(invF-2), but they are transcribed in opposite directions. However, invH expression did not influence P(invF-2) activity. This suggests that independent regulation of the two invF promoters allows Salmonella to respond quickly to environmental changes.
Microbial Pathogenesis 03/2012; 52(6):359-66. · 1.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A Gram-staining-positive, strictly aerobic, spherical-shaped, non-motile red-pigmented bacterial strain MK03T, was isolated from a soil sample. A polyphasic approach was applied to study the taxonomic position of strain MK03T. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MK03T belonged to the clade formed by members of the genus Deinococcus in the family Deinococcaceae. The G+C content of the genomic DNA was 64.5 mol %. Strain MK03T showed highest 16S rRNA gene sequence similarity with Deinococcus aerolatus 5516T-9T (97.4 %), Deinococcus marmoris AA-63 T (97.2 %), Deinococcus radiopugnans ATCC19172 T (97.2 %.) and Deinococcus saxicola AA-1444T (96.9 %), whereas, sequence similarity to other Deinococcus species was less than 96.0 %. The chemotaxonomic characteristics of strain MK03T showed typical features of the genus Deinococcus, with the presence of predominant respiratory quinone as MK-8, the major fatty acids (>12 %) C16:1ω7c, C15:1ω6c, C16:0 and C15:0, ornithine as diamino acid in the cell wall peptidoglycan and resistance to gamma radiation [D10 (dose required to reduce the bacterial population by 10-fold) >9 kGy]. The low DNA-DNA relatedness values (34.5±1.5-11.6±2.4 %) indicated that strain MK03T represented a distinct species that was separated from other closely related type strains in the genus Deinococcus. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MK03T is described as a novel species of the genus Deinococcus, for which the name Deinococcus humi sp. nov. is proposed. The type strain is MK03T (=KCTC 13619T = JCM 17915T).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 01/2012; · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bacterial cell-to-cell communication, termed quorum sensing (QS), controls bacterial behavior by using various signal molecules. Despite the fact that the LuxS/autoinducer-2 (AI-2) QS system is necessary for normal expression of Salmonella pathogenicity island-1 (SPI-1), the mechanism remains unknown. Here, we report that the LsrR protein, a transcriptional regulator known to be involved in LuxS/AI-2-mediated QS, is also associated with the regulation of SPI-1-mediated Salmonella virulence. We determined that LsrR negatively controls SPI-1 and flagella gene expressions. As phosphorylated AI-2 binds to and inactivates LsrR, LsrR remains active and decreases expression of SPI-1 and flagella genes in the luxS mutant. The reduced expression of those genes resulted in impaired invasion of Salmonella into epithelial cells. Expression of SPI-1 and flagella genes was also reduced by overexpression of the LsrR regulator from a plasmid, but was relieved by exogenous AI-2, which binds to and inactivates LsrR. These results imply that LsrR plays an important role in selecting infectious niche of Salmonella in QS dependent mode.
PLoS ONE 01/2012; 7(5):e37059. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A Gram-stain-positive, strictly aerobic, spherical, non-motile red-pigmented bacterial strain, designated MJ27(T), was isolated from a sludge sample of the Daejeon sewage disposal plant in South Korea. A polyphasic approach was used to study the taxonomic position of strain MJ27(T). Strain MJ27(T) shared highest 16S rRNA gene sequence similarity with Deinococcus grandis DSM 3963(T) (98.8 %), Deinococcus caeni Ho-08(T) (97.5 %) and Deinococcus aquaticus PB314(T) (96.6 %.); levels of sequence similarity with the type strains of other Deinococcus species were less than 96.0 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ27(T) belonged to the clade formed by members of the genus Deinococcus in the family Deinococcaceae. The G+C content of the genomic DNA of strain MJ27(T) was 67.6 mol%. The chemotaxonomic characteristics of strain MJ27(T) were typical of members of the genus Deinococcus, with MK-8 as the predominant respiratory quinone, C(16:1)ω7c, C(15:1)ω6c, C(16:0) and C(15:0) as major fatty acids (>12 %), ornithine as the diamino acid in the cell-wall peptidoglycan and resistance to gamma radiation [D(10) (dose required to reduce the bacterial population by tenfold) >9 kGy]. The low levels of DNA-DNA relatedness reported here (5.3±1.5-29.2±2.3 %) indicate that strain MJ27(T) represents a species that is separate from its closest relatives in the genus Deinococcus. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ27(T) is considered to represent a novel species of the genus Deinococcus, for which the name Deinococcus daejeonensis sp. nov. is proposed. The type strain is MJ27(T) ( = KCTC 13751(T) = JCM 16918(T)).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 07/2011; 62(Pt 6):1265-70. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Both Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. Typhimurium) have a tdc operon that encodes enzymes involved in a metabolic pathway for the degradation of L-serine and L-threonine. However, S. Typhimurium does not have the tdcR gene, which is a positive regulator in E. coli. In the present study, transcriptional analysis revealed that tdcA expression in E. coli is higher under anaerobic than aerobic growth conditions, but the opposite is true in S. Typhimurium. Interestingly, a tdcR mutant strain of E. coli showed a similar expression pattern to that observed in S. Typhimurium and was also induced by anaerobic shock. These results suggest that the induction of tdcA expression by anaerobic conditions is observable when tdcA expression is low owing to the absence of TdcR.
Journal of Microbiology and Biotechnology 03/2011; 21(3):252-5. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNAdamaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.
Journal of Microbiology and Biotechnology 12/2010; 20(12):1637-46. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Salmonella tdc operon encodes enzymes belonging to a metabolic pathway that degrades L-serine and L-threonine. The upregulation of the tdc operon and increased virulence of Salmonella grown under oxygen-limiting conditions prompted us to investigate the role of the tdc operon in the pathogenesis of Salmonella Typhimurium. A Salmonella strain carrying a null mutation in tdcA, which encodes the transcriptional activator of the tdc operon, was impaired in mice infected intraperitoneally with the bacterium. In addition, the Salmonella tdcA mutant showed reduced replication compared with the parental strain in cultured animal cells, although their growth rates were similar in various culture media. To understand the function of TdcA in pathogenesis, we performed two-dimensional gel electrophoresis and found that flagellar and PhoP-regulated proteins were affected by the tdcA mutation. The results of beta-galactosidase assays and FACS analysis showed that, among the four PhoP-dependent genes tested, the expression of ssaG, which is located in Salmonella pathogenicity island 2 (SPI2), was reduced in the tdcA mutant, especially in the intracellular environment of macrophages. Taken together, our data suggest that tdcA plays an important role in the pathogenesis of Salmonella.
Molecules and Cells 05/2010; 29(5):509-17. · 2.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously observed that the transcription of some flagellar genes decreased in Salmonella Typhimurium tdcA mutant, which is a gene encoding the transcriptional activator of the tdc operon. Since flagella-mediated bacterial motility accelerates the invasion of Salmonella, we have examined the effect of tdcA mutation on the invasive ability as well as the flagellar biosynthesis in S. Typhimurium. A tdcA mutation caused defects in motility and formation of flagellin protein, FliC in S. Typhimurium. Invasion assays in the presence of a centrifugal force confirmed that the defect of flagellum synthesis decreases the ability of Salmonella to invade into cultured epithelial cells. In addition, we also found that the expression of Salmonella pathogenicity island 1 (SPI1) genes required for Salmonella invasion was down-regulated in the tdcA mutant because of the decreased expression of fliZ, a positive regulator of SPI1 transcriptional activator, hilA. Finally, the virulence of a S. Typhimurium tdcA mutant was attenuated compared to a wild type when administered orally. This study implies the role of tdcA in the invasion process of S. Typhimurium.
Molecules and Cells 09/2009; 28(4):389-95. · 2.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The taxonomic positions of two environmental isolates from South Korea were established using a combination of genotypic and phenotypic data. The organisms, designated PB314(T) and Ho-08(T), were Gram-negative, rod-shaped and non-spore-forming and had chemotaxonomic properties consistent with their classification in the genus Deinococcus 16S rRNA gene tree, the highest sequence similarities being shown to the type strains of Deinococcus grandis (96.3-96.7 %) and Deinococcus indicus (96.3-96.4 %). The isolates shared relatively high 16S rRNA gene sequence similarity (98.1 %) but had a DNA-DNA relatedness value of only 22 %. Chemotaxonomic data revealed that both strains possess quinone system MK-8 as the predominant compound, C(16 : 1)omega7c and C(16 : 0) as major fatty acids and ornithine as a diamino acid in the peptidoglycan structure, corroborating our assignment of the strains to the genus Deinococcus. The results of phylogenetic analyses based on 16S rRNA gene sequences, DNA-DNA relatedness values and physiological and biochemical tests clearly demonstrated that the two strains represent distinct species. On the basis of these data, two novel species, Deinococcus aquaticus sp. nov. (type strain PB314(T) =KCTC 12552(T) =NBRC 101311(T)) and Deinococcus caeni sp. nov. (type strain Ho-08(T) =KCTC 12553(T) =NBRC 101312(T)), are proposed.
International journal of systematic and evolutionary microbiology 11/2008; 58(Pt 10):2348-53. · 2.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Efficient expression of the Salmonella Typhimurium tdcABCDEG operon involved in the degradation of Lserine and L-threonine requires TdcA, the transcriptional activator of the tdc operon. We found that the tdcA gene was transiently activated when bacterial growth condition was changed from aerobic to anaerobic, but this was not observed if Salmonella was grown anaerobically from the beginning of the culture. Expression kinetics of six tdc genes after anaerobic shock demonstrated by a real-time PCR assay showed that the tdcCDEG genes were not induced in tdcA mutant but tdcB maintained its inducibility by anaerobic shock even in the absence of tdcA, suggesting that an additional unknown transcriptional regulation may work for the tdcB expression. We also investigated the effects of nucleoid-associated proteins by primer extension analysis and found that H-NS repressed tdcA under anaerobic shock conditions and fis mutation delayed the peak expression time of the tdc operon. DNA microarray analysis of genes regulated by TdcA revealed that the genes involved in Nacetylmannosamine, maltose, and propanediol utilization were significantly induced in a tdcA mutant. These findings suggest that Tdc enzymes may play a pivotal role in energy metabolism under a sudden change of oxygen tension.
Journal of Microbiology and Biotechnology 07/2008; 18(6):1024-32. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The global regulator Mlc is a repressor of several genes and operons that are involved in sugar uptake and metabolism. A Salmonella enterica serovar Typhimurium mlc mutant showed reduced levels of invasion and cytotoxicity compared to the wild-type, and exhibited reduced expression levels of hilD, hilA and invF, which are regulatory genes in the Salmonella pathogenicity island 1 (SPI1). However, the effects of Mlc on hilD expression and bacterial invasiveness were not seen in the hilE mutant, and hilE expression was increased in the mlc mutant, which suggests that Mlc exerts positive effects on the expression of SPI1 genes by reducing the expression of HilE, which is known to down-regulate the expression of SPI1 genes through direct interaction with HilD. We found that the two known promoters of hilE were not modulated by Mlc, and we identified a third promoter, designated P3, which was repressed by Mlc. The gel mobility shift assay and footprinting analysis revealed that Mlc repressed hilE in a direct manner by binding to two distinct sites in the hilE P3 promoter region. The specific down-regulation of hilD observed in the presence of Mlc regulon-inducible sugars, such as glucose and mannose, could not be detected in the mlc mutant. Based on these results, we propose that Mlc functions to sense the availability of sugars and is linked to virulence gene regulation by its ability to control hilE expression in Salmonella.
Nucleic Acids Research 02/2007; 35(6):1822-32. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Salmonella pathogenicity island 2 (SPI2) encodes a type III secretion system (TTSS) necessary for bacterial survival and replication in intracellular environment of host cells. SPI2 genes are transcribed preferentially after Salmonella enters the host cells. Transcriptional regulation of ssaG encoding the component of SPI2-TTSS apparatus was studied in vivo and in vitro. Fis, one of the major components of bacterial nucleoid, activated the stationary phase-specific expression of ssaG when Salmonella was grown in LB media. Gel-shift and footprinting analysis showed Fis bound to four distinct sites of the ssaG promoter region with different affinities. All four Fis-binding sites were required for timely transcription activation of ssaG after Salmonella entered macrophage cells. Gentamicin protection experiments using bacteria grown to stationary phase prior to infection showed that the ability of the fis mutant strain to replicate within the RAW264.7 macrophage cells was lower than the wild type. These observations confirm that Fis plays an important role in regulations of SPI2 as well as SPI1 for an efficient regulation of the virulence genes.
Microbial Pathogenesis 08/2006; 41(1):33-42. · 1.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To study the radiosensitivity of DNA segments at the open reading frame (gene) level, real-time PCR was used to analyze DNA damages induced by ionizing radiation. After irradiation (1, 3 and 5 kGy) of genomic DNA purified from Salmonella typhimurium, real-time PCR based on SYBR Green fluorescence and melting temperature was performed using various primer sets targeting the rfbJ, rfaJ, rfaB, hilD, ssrB, pipB, sopD, pduQ, eutG, oadB, ccmB and ccmA genes. The ccmA and ccmB genes, which existed as two copies on the chromosome and had a high GC content ( approximately 70%), showed much lower radiosensitivities than the other genes tested, particularly at 5 kGy; this distinctive feature was seen only when the genes were located on the chromosome, regardless of copy number. Our results reinforce the concept that gene sensitivity to ionizing radiation depends on the base composition and/or the spatial localization of the gene on the chromosome.
Radiation Research 05/2006; 165(4):430-7. · 2.68 Impact Factor
-
Miryoung Song,
Hyun-Ju Kim,
Eun Young Kim,
Minsang Shin,
Hyun Chul Lee,
Yeongjin Hong,
Joon Haeng Rhee,
Hyunjin Yoon,
Sangryeol Ryu, Sangyong Lim,
Hyon E Choy
[show abstract]
[hide abstract]
ABSTRACT: We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well defined standard growth condition of Luria-Bertani medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, the two-component regulatory genes hilC/hilD, sirA/barA, and ompR, which are known to modulate expression from the hilA promoter (hilAp) under so-called "inducing conditions" (Luria-Bertani medium containing 0.3 m NaCl without aeration), acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by the addition of serine hydroxamate did not induce hilAp in a RelA(+) SpoT(+) strain suggested that, in addition to ppGpp, some other alteration accompanying entry into the stationary phase might be necessary for induction. It is speculated that during the course of infection Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, which allows expression of Salmonella virulence genes, including SPI1 genes.
Journal of Biological Chemistry 09/2004; 279(33):34183-90. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Salmonella enterica serovar Typhimurium is an enteric pathogen and a principal cause of gastroenteritis in humans. The factor-for-inversion stimulation protein (Fis) is known to play a pivotal role in the expression of Salmonella pathogenicity island (SPI)-1 genes in addition to various cellular processes such as recombination, replication, and transcription. In order to understand Fis function in pathogenicity of Salmonella, we performed two-dimensional gel electrophoresis and identified proteins whose expression pattern is affected by Fis using mass spectrometry. The results revealed various proteins that can be grouped according to their respective cellular functions. These groups include the genes involved in the metabolism of sugar, flagella synthesis, translation, and SPI expression. Changes in SPI expression suggest the possibility that regulation of genes in SPI-2 as well as SPI-1 is affected by Fis.
FEMS Microbiology Letters 10/2003; 226(2):391-6. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold (CT) increased 1–1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared CT values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3–3.3-fold underestimation of bacterial counts with respect to irradiation dose.
Radiation Physics and Chemistry.
-
[show abstract]
[hide abstract]
ABSTRACT: The principle benefit of food irradiation is the reduction of food-borne bacteria in food products. However, the microbiological safety with respect to increased virulence of surviving pathogens after irradiation remains an important issue with regard to the effectiveness of food irradiation. In this study, the transcriptional changes of virulence genes of Salmonella and Vibrio spp. after γ radiation were investigated by real-time PCR (RT-PCR). Samonella typhimurium is dependent upon the products of a large number of genes located within Salmonella pathogenicity islands (SPI) on the chromosome. The expressions of seven genes including four SPI genes, hilD, ssrB, pipB, and sopD, were measured at 1 h after 1 kGy irradiation. Compared with non-irradiated controls, the expression of hilD encoded within SPI1 and sopD encoding SPI1-related effector proteins was reduced about 4- and 16-fold, respectively. The expressions of Vibrio toxin genes, vvhA, ctxA, and tdh, were also monitored during the course of a growth cycle after re-inoculation of irradiated Vibrio spp. (0.5 and 1.0 kGy). The expressions of Vibrio toxin genes tested did not increase compared with non-irradiated counterparts. Results from this study indicate that γ radiation is much more likely to reduce the virulence gene expression of surviving pathogens.
Radiation Physics and Chemistry. 76:1763-1766.
