16S rRNA Levels Research Articles

Cohnella ferri sp. nov. A Novel Member of the Genus Cohnella Isolated from Haematite Ore

The taxonomic position of a Gram-positive, endo-spore forming bacterium isolated from a haematite ore sample was analyzed by a polyphasic approach. The strain designated as HIO-4(T) matched most of the phenotypic and chemical characteristics of the genus Cohnella and represents a novel species. The sequence of the almost complete 16S rRNA (1489 bases) was compared with those of previously studied Cohnella type strains and confirmed that the strain belongs to the genus Cohnella. Strain HIO-4(T) differs from all other species of Cohnella by at least 3.9% at the 16S rRNA level and the moderately related species are Cohnella phaseoli (96.1%) and Cohnella yongneupensis (96.1%), respectively. Predominant polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE); few unknown phospholipids, mannose containing lipid, aminophospholipid and aminophosphoglycolipids. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain HIO-4(T) with its phylogenetic relatives and suggest that the strain HIO-4(T) should be recognized as a novel species, for which the name Cohnella ferri sp. nov. is proposed. The type strain is HIO-4(T) (=MTCC 8365(T)=JCM 16139(T)).

Dynamic transition of microbial communities in response to acidification in fixed-bed anaerobic baffled reactors (FABR) of two different flow directions

Two five-compartment fixed-bed anaerobic baffled reactors (FABRs) were operated under deteriorative and stable conditions. The FABRs were identical except for flow direction: one was horizontal (H-reactor) and the other was vertical (V-reactor). The microbial community dynamics in 1, 3 and 5 compartments were analyzed using denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone library screening and quantitative PCR. After start-up, the Methanomicrobiales were typically dominant in adhering sludge of 5th compartments of two reactors. Because methanogenesis mainly occurred in the latter compartment, Methanomicrobiales were likely to play important roles in FABRs. FABRs recovered from performance deterioration very quickly. Meanwhile, methanogens and dominant methanogens greatly increased in every compartment of two reactors. Our results indicated that 16S rRNA levels of methanogens in the adhering sludge were higher than those in the deposited sludge and the adhering fraction of V-reactor held up more acid-resistant bacteria and methanogens than that of H-reactor.

Nop6, a component of 90S pre-ribosomal particles, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ribosome biogenesis requires, in addition to rRNA and ribosomal proteins, a myriad of small nucleolar RNAs (snoRNAs) and over two hundred protein trans-acting factors. There are protein trans-acting factors predicted to participate in ribosome biogenesis that have not been so far characterized. Here, we report the functional analysis of the Nucleolar protein 6 (Nop6) in ribosome biogenesis. Our results show that Nop6 is needed for optimal 40S ribosomal subunit biogenesis. Deletion of NOP6 leads to an appropriate 20% reduction in 18S rRNA levels and therefore in 40S ribosomal subunits. This is due to mild inhibition of pre-rRNA processing at cleavage site A2. Tandem affinity purification followed by mass spectrometry and northern blot analyses indicate that Nop6 is a component of 90S pre-ribosomal particles. rDNA chromatin immunoprecipitation experiments and analysis of the intracellular localisation of Nop6-eGFP after in vivo shut down of pre-rRNA transcription strongly suggest that Nop6 binds to the pre-rRNA early during transcription. Genetic data suggest that Nop6 and the snoRNA snR57 both interact similarly with the protein trans-acting factor Nep1. It has been proposed that snR57 and Nep1 participate in a pre-rRNA conformational switch that allows the proper assembly of 40S ribosomal protein S19. Our results strongly suggest that the role Nop6 might have in this conformational switch is independent of snR57.

Dyskerin is required for tumor cell growth through mechanisms that are independent of its role in telomerase and only partially related to its function in precursor rRNA processing

Dyskerin is an essential nucleolar protein required for the biogenesis of ribonucleoproteins that incorporate H/ACA RNAs. Through binding to specific H/ACA RNAs, dyskerin exerts most of its influence in the cell. To that end, dyskerin is a core component of the telomerase complex and is required for normal telomere maintenance. Dyskerin is also required for post-transcriptional processing of precursor rRNA. Germline dyskerin mutations increase cancer susceptibility. Conversely, wild-type dyskerin is usually over-expressed and not mutated in sporadic cancers. However, the contributions of dyskerin to sporadic tumorigenesis are unknown. Described herein, we demonstrate that acute loss of dyskerin function by RNA interference significantly reduced steady-state levels of H/ACA RNAs, disrupted the morphology and inhibited anchorage-independent growth of telomerase-positive and telomerase-negative human cell lines. Unexpectedly, dyskerin depletion only transiently delayed rRNA maturation but with no appreciable effect on the levels of total 18S or 28S rRNA. Instead, while rRNA processing defects typically trigger p53-dependent G1 arrest, dyskerin-depleted cells accumulated in G2/M by a p53-independent mechanism, and this was associated with an accumulation of aberrant mitotic figures that were characterized by multi-polar spindles. Telomerase activity and the rate of rRNA processing are typically increased during neoplasia. However, our cumulative findings indicate that dyskerin contributes to tumor cell growth through mechanisms which do not require the presence of cellular telomerase activity, and which may be only partially dependent upon the protein's role in rRNA processing. These data also reinforce the notion that loss and gain of dyskerin function may play important roles in tumorigenesis.

Effects on mitochondrial transcription of manipulating mTERF protein levels in cultured human HEK293 cells

BackgroundBased on its activities in vitro, the mammalian mitochondrial transcription termination factor mTERF has been proposed to regulate mitochondrial transcription by favouring termination at its high-affinity binding immediately downstream of the rDNA segment of mitochondrial DNA, and initiation selectively at the PH1 site of the heavy-strand promoter. This defines an rDNA transcription unit distinct from the 'global' heavy-strand transcription unit initiating at PH2. However, evidence that the relative activities of the two heavy-strand transcription units are modulated by mTERF in vivo is thus far lacking.ResultsTo test this hypothesis, we engineered human HEK293-derived cells for over-expression or knockdown of mTERF, and measured the steady-state levels of transcripts belonging to different transcription units, namely tRNALeu(UUR) and ND1 mRNA for the PH2 transcription unit, and tRNAPhe plus 12S and 16S rRNA for the PH1 transcription unit. The relative levels of 16S rRNA and ND1 mRNA were the same under all conditions tested, although mTERF knockdown resulted in increased levels of transcripts of 12S rRNA. The amount of tRNAPhe relative to tRNALeu(UUR) was unaffected by mTERF over-expression, altered only slightly by mTERF knockdown, and was unchanged during recovery from ethidium bromide-induced depletion of mitochondrial RNA. mTERF overexpression or knockdown produced a substantial shift (3-5-fold) in the relative abundance of antisense transcripts either side of its high-affinity binding site.ConclusionsmTERF protein levels materially affect the amount of readthrough transcription on the antisense strand of mtDNA, whilst the effects on sense-strand transcripts are complex, and suggest the influence of compensatory mechanisms.

Mitomycin C Inhibits Ribosomal RNA: A NOVEL CYTOTOXIC MECHANISM FOR BIOREDUCTIVE DRUGS

Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds.

Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

BackgroundReverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results.MethodsWe assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values.ResultsThis RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination.ConclusionThis study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

Deregulation of oncogene-induced senescence and p53 translational control in X-linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X-linked dyskeratosis congenita (X-DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene-induced senescence (OIS). Here, we show that during OIS, a switch between cap- and internal ribosome entry site (IRES)-dependent translation occurs. During this switch, an IRES element positioned in the 5'untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1(m) cells, p53 IRES-dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X-DC human patient cells in which similar impairments in p53 IRES-dependent translation are observed. Importantly, re-introduction of wild-type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.

Abstract 5010: Nucleophosmin (NPM) is univerally deregulated in acute promyelocytic leukemia

Abstract NPM is a multifunctional nucleolar phosphoprotein with roles in ribosome biogenesis, centrosome duplication, p53 response and DNA repair, and is commonly mutated in Acute myelogenous leukemia (AML). NPM is juxtaposed with retinoic acid receptor α (RARα[[Unsupported Character - [[Unsupported Character - &amp;#61481;]]]]) in Acute Promyelocytic Leukemia (APL), raising the possibility that NPM functions are disrupted in this leukemia. We sought to determine the extent of NPM deregulation in the U937-NPM-RARα+ cell line model, and to extend this analysis more generally to APL. Immunofluorescent microscopy (IF) demonstrated abnormal distribution of NPM in patient-derived APL cells carrying NPM-RARα or PML-RARα, as well as cell lines expressing X-RARα: NPM was distributed into abnormally large aggregates within the nucleus and/or throughout the cytoplasm. NPM distribution reverted to normal after 48 hr of treatment with 1.0 μM all-trans retinoic acid (ATRA). NPM protein levels were also significantly increased in X-RARα+ cell lines. This may by due to an alteration at the protein level, as NPM mRNA expression was unaffected by X-RARα, and no mutations were detected in the NPM locus. Indeed, we found that NPM protein half-life was increased in U937 cells expressing X-RARα. These data suggested a potential disruption in nucleolar architecture in APL cells. Alterations in size and number of nucleolar organizing regions (NORs) in NB4 and U937-X-RARα cells were evident, when compared to U937 controls. Defects in nucleolar organization may lead to altered ribosome biogenesis, protein synthesis, as well as cell size and proliferation. Ribosomal RNA precursor expression was found to be increased significantly in U937-NPM-RARα cells, compared to controls. Relative cell volume, assessed by a flow cytometric assay, was moderately increased, by approximately 10% in NPM-RARα+ cells, while cell proliferation rates were significantly increased, and doubling time decreased, compared to U937 controls. Analysis of protein synthesis rates in U937-NPM-RARα+ cells indicated that the incorporation of a fluorescent Methionine analogue was twice that in control U937 cells. Finally, in order to determine the applicability of our in vitro results to APL patients, we analyzed pre-rRNA and 18S rRNA expression in bone marrow RNA extracted at diagnosis from 16 APL patients (10 BCR1/2 and 6 BCR3), in order to determine whether elevated nucleolar function was found in APL. 10/16 APLs had elevated 18S rRNA levels (&amp;gt;1.5-fold compared to normal BM); 8/16 had similarly elevated pre-rRNA levels. Overall, pre-rRNA levels were elevated an average of 2-fold compared to normal controls, while 18S rRNA levels were elevated an average 1.8-fold (p &amp;lt; 0.05 for both comparisons). We therefore present the first evidence that NPM may be universally deregulated in APL, leading to defective NPM function within the leukemic cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5010.

Abstract 4112: Lysophosphatidic acid up-regulates expression of TNF-alpha in human liver cancer cell lines

Abstract Background: Lysophosphatidic acid (LPA) is an important intercellular signaling molecule involved in a number of physiologic and pathophysiological processes. Elevated serum LPA was reported to be associated with liver fibrosis in patients with chronic hepatitis C. We have previously demonstrated that the enzyme mediated LPA generation- autotaxin(ATX) was overexpressed in hepatitis and hepatitis associated HCC. But the exact mechanism of the enhanced LPA signaling contributed to the initiation or progression of tumorigenesis in human liver cancer remains unclear. Methods: Human liver cancer cell lines Hep3B, HepG2 and Huh7 were serum-starved overnight and stimulated with LPA (18:1, Avanti Polar-Lipids, Inc., Alabaster, AL, USA) by different doses (0, 2.5, 5, 10 and 20 uM) and different time intervals (3, 8 and 16 hours). RNA was extracted using Qiagen kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Quantitative real-time PCR was applied to determine the expression of interleukin 1, beta (IL-1B), IL-6, tumor necrosis factor alpha(TNF-alpha), transforming growth factor alpha 1 (TGF-alpha1) and transforming growth factor beta1 (TGF-beta1) using standard commercially available TaqMAN probes (Applied Biosystems, Foster City, CA). The amount of target gene was normalized to the internal standard 18S rRNA (Hs99999901_s1) levels and reported as a relative value. Results: TNF-alpha mRNA level was rapidly induced more than 3-fold after stimulating with 2.5 uM LPA for 3 hours in Hep3B cells (P&amp;lt;0.01), but this induction did not show significant change in spite of increasing treatment time or LPA doses. The curves of TNF-alpha expression in Huh7 and HepG2 cells were nearly identical to that of Hep3B cells, Treatment with 2.5uM LPA for 3 hours induced more than 5- and 2- fold TNF-alpha mRNA in Huh7 and HepG2 cells respectively(p&amp;lt;0.001). Different dose or treatment time did not result in a significant increase of induction of TNF-alpha mRNA in these two liver cancer cell lines. Interestingly, LPA treatment had no effect on the mRNA expression of TGF-alpha1, TGF-beta1, IL-1 or IL-6 in these three liver cancer cell lines in spite of increasing LPA doses or time intervals. Conclusions: Our study demonstrates that LPA transcriptionally activates TNF-alpha expression in human liver cancer cell lines. TNF-alpha is known to induce hepatocytes apoptosis and is one of the few critical factors that prime liver regeneration. LPA-induced TNF-alpha production in liver cancer cells may be important not only for tumor behavior, but also has potential damage to normal tissues which in turn fuel the liver tumor progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4112.

Evolutionarily Conserved Function of RRP36 in Early Cleavages of the Pre-rRNA and Production of the 40S Ribosomal Subunit

Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

Stress-Induced Hsp70 Gene Expression and Inactivation of Cryptosporidium parvum Oocysts by Chlorine-Based Oxidants

Our research on the mechanisms of action of chlorine-based oxidants on Cryptosporidium parvum oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on C. parvum oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of beta-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.

Correlated Levels of mRNA and Soma Size in Single Identified Neurons: Evidence for Compartment-specific Regulation of Gene Expression

In addition to the overall complexity of transcriptional regulation, cells also must take into account the subcellular distribution of these gene products. This is particularly challenging for morphologically complex cells such as neurons. Yet the interaction between cellular morphology and gene expression is poorly understood. Here we provide some of the first evidence for a relationship between neuronal compartment size and maintenance of mRNA levels in neurons. We find that single-cell transcript levels of 18S rRNA, GAPDH, and EF1-alpha, all gene products with primary functions in the cell soma, are strongly correlated to soma size in multiple distinct neuronal types. Levels of mRNA for the K+ channel shal, which is localized exclusively to the soma, are negatively correlated with soma size, suggesting that gene expression does not simply track positively with compartment size. Conversely, levels of beta-actin and beta-tubulin mRNA, which are major cytoskeletal proteins of neuronal processes, do not correlate with soma size, but are strongly correlated with one another. Additionally, actin/tubulin expression levels correlate with voltage-gated ion channels that are uniquely localized to axons. These results suggest that steady-state transcript levels are differentially regulated based on the subcellular compartment within which a given gene product primarily acts.

The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.

Thermus arciformis sp. nov., a thermophilic species from a geothermal area

Two aerobic, Gram-negative, non-motile, non-sporulating, yellow-pigmented bacteria, strains TH92(T) and TH91, were isolated from a hot spring located in Laibin, Guangxi, in the south-eastern geothermal area of China. The isolates grew at 40-77 degrees C (optimally at 70 degrees C) and at pH 6.0-9.5 (optimally at pH 7.5-8.0). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicated that the new isolates represented a novel species of the genus Thermus with closest affinity to Thermus aquaticus, Thermus igniterrae and Thermus thermophilus. Compared with their closest relatives, strains TH92( T) and TH91 were able to assimilate a wider range of carbohydrates, amino acids and organic acids as sole carbon sources for growth, such as lactose and melibiose. The new isolates had lower combined levels of C(16 : 0 ) and iso-C(16 : 0) compared with their closest relatives. On the basis of polyphasic taxonomic characterization, strains TH92(T) and TH91 are considered to represent a single novel species of the genus Thermus, for which the name Thermus arciformis sp. nov. is proposed. The type strain is TH92(T) (=CGMCC 1.6992(T) =JCM 15153(T)).

An RNA spiking method demonstrates that 18S rRNA is regulated by progesterone in the mouse uterus

Identifying suitable housekeeping genes for quantitative RT-PCR in the uterus is problematic, as this tissue undergoes significant structural and functional alterations during the oestrous cycle and pregnancy in response to circulating hormones. The suitability of 18S rRNA as a housekeeping gene in mouse uterus was investigated by introducing an 'RNA spike' standard into the reverse transcription reaction. 18S rRNA levels increased by Day 4 of pregnancy and after progesterone administration in ovariectomized mice. We conclude that 18S rRNA is not a suitable housekeeping gene for quantitative RT-PCR analysis in progesterone-responsive tissues, and the RNA spiking method provides a suitable alternative.

Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5' tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055(T). Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.

&lt;I&gt;MYP&lt;/I&gt; gene expressions at transcription level in different stages of gonad of sea urchin &lt;I&gt;Strongylocentrotus intermedius&lt;/I&gt; and hybrids

MYP (Major yolk protein) gene expression at transcription level in different stages of gonad of sea urchin Strongylocentrotus intermedius and hybrids (S. intermediusfemale symbolxS. nudusmale symbol) was analyzed by real-time RT-PCR. Based on normalization with 18S rRNA levels, the comparative quantities of MYP expression were determined. The expression of MYP gene in gonad showed little difference between female and male. MYP gene expression was decreased rapidly in the gonad of S. intermedius at different stages, and slowly in hybrids. The comparative quantities of MYP expression in the gonads of S. intermedius were decreased from 44.55% to 9.59% in female and from 41.17% to 1.83% in male at different stages. The comparative quantities of MYP expression in the gonads of the hybrids were decreased from 37.66% to 19.22% in female and from 36.66% to 12.55% in male at different stages. The results indicated that the difference of MYP expression was correlated with the variation caused by hybridization.

Enrichment of a Mixed Bacterial Culture with a High Polyhydroxyalkanoate Storage Capacity

Polyhydroxyalkanoates (PHAs) are microbial storage polymers that attract interest as bioplastics. PHAs can be produced with open mixed cultures if a suitable enrichment step based on the ecological role of PHA is used. An acetate-fed sequencing batch reactor operated with 1 day biomass residence time and with feast-famine cycles of 12 h was used to enrich a mixed culture of PHA producers. In subsequent fed-batch experiments under growth limiting conditions, the enriched mixed culture produced PHA up to a cellular content of 89 wt % within 7.6 h (average rate of 1.2 g/g/h). The PHA produced from acetate was the homopolymer polyhydroxybutyrate. The culture was dominated by a Gammaproteobacterium that showed little similarity on 16S rRNA level with known bacteria (<90% sequence similarity). The mixed culture process for PHA production does not require aseptic conditions. Waste streams rather than pure substrates could be used as raw materials.

Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses

Four bacterial strains, designated ST18(T), HM244, HM250 and DI49, were isolated from the fresh faeces of four thoroughbred horses in Japan. Cells were Gram-positive, strictly anaerobic, catalase-negative, non-spore-forming, non-motile rods that occurred in chains. They were placed in the same subcluster based on 16S rRNA gene sequence analysis, phenotypic characteristics and levels of DNA-DNA relatedness. Their DNA G+C content ranged from 36 to 38 mol%. Lactobacillus catenaformis, Lactobacillus vitulinus and Catenibacterium mitsuokai belong to cluster XVII of the Clostridium subphylum. Strain ST18(T) was most closely related to L. catenaformis ATCC 25536(T) in the phylogenetic tree, but these strains shared only 89.9 % (1336/1486 bp) 16S rRNA gene sequence similarity. L. catenaformis, L. vitulinus and C. mitsuokai are homofermentative bacteria, whereas ST18(T) produced CO(2) from glucose. Whereas the cell-wall peptidoglycan type of L. catenaformis and L. vitulinus wasL-Lys-L-Ala(3), that of C. mitsuokai and the subgroup represented by ST18(T) was A1gamma (L-Ala-D-Glu-meso-diaminopimelic acid). On the basis of 16S rRNA gene sequence divergence of more than 10 % from L. catenaformis as well as phenotypic characteristics, strains ST18(T), HM244, HM250 and DI49 are considered to represent a novel species of a new genus belonging to the Clostridium subphylum cluster XVII, for which the name Sharpea azabuensis gen. nov., sp. nov. is proposed. The type strain of Sharpea azabuensis is ST18(T) (=JCM 14210(T) =DSM 18934(T)).