Unique glycine-activated riboswitch linked to glycine–serine auxotrophy in SAR11

The genome sequence of the marine bacterium ' Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that ' Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 μM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 × 10⁶ cells ml⁻¹ to 8.16 × 10⁶ cells ml⁻¹ ( R ² =  0.992). Serine was capable of substituting for glycine at 1.5 μM. ' Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that ' Cand. P. ubique' is therefore a glycine–serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.     

Instability of the L6 gene for rust resistance in flax is correlated with the presence of a linked Ac element

In a programme aimed at tagging rust-resistance genes in flax with the maize transposable element Ac , a primary transformant of a line called 'Forge' that is homozygous for four rust-resistance genes, L ⁶, M, N and P ², was identified that possessed 10 copies of the Ac element, one of which was linked (29 map units) to L ⁶. Descendants of this plant, which had from 8 to 15 copies of Ac , were crossed to a rust-susceptible line and the progeny screened for rust-susceptible mutants. When the Ac linked to L ⁶ was present in the parent, a high frequency of L ⁶ mutants was observed (29 mutants in 30 575). By contrast, when this Ac was absent, no such mutants were observed in 9258 progeny. The background frequency of L ⁶ mutants was low (five in 124 088). A detailed analysis was made of the first 11 L ⁶ mutants recovered from parents carrying the L ⁶-linked Ac element. While none of the mutants possessed a tagged resistance gene, all lacked an RFLP marker closely linked to L ⁶, suggesting that deletions were responsible for loss of the L ⁶ specificity. In many of the mutants, one or more RFLP markers in the vicinity of the linked Ac were also absent. These findings suggest that the linked Ac may be inducing chromosome breakage.     

Characterization of the dual start motif of a class II holin gene

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S  ^λ, has a dual start motif and encodes not only the effector holin, S^λ105, but also an inhibitor, S^λ107, with a Met–Lys … extension at the terminus. The prototype class II holin gene of phage 21, S  ²¹, begins with the motif Met–Lys–Ser–Met … , and a potential RNA secondary structure overlaps the Shine–Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S  ²¹, S²¹71 and S²¹68; (ii) the shorter product is required for lysis; (iii) the longer product, S²¹71, inhibits S  ²¹ function; and (iv) the Lys-2 residue is important for the inhibitor function. Moreover, the RNA stem–loop structure is involved in the downregulation of S²¹71 synthesis. However, our results suggest that, in S  ²¹, different segments of the single consensus Shine–Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn²⁺. In the presence of 0.5 mM EGTA, only the parental strain was able to take up ⁶⁵Zn²⁺. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn²⁺ concentrations, the znu mutants took up more ⁶⁵Zn²⁺ than the parental strain. The high-affinity ⁶⁵Zn²⁺ uptake was repressed by growth in the presence of 10 μM Zn²⁺. A znuA–lacZ operon fusion was repressed by 5 μM Zn²⁺ and showed a more than 20-fold increase in β-galactosidase activity when Zn²⁺ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn²⁺-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity ⁶⁵Zn²⁺ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans

We have identified a novel 399 bp repetitive DNA element (which we designate beta  ) 9 bp upstream of a seryl-tRNA_CAG gene in the genome of Candida albicans . There are two copies of the seryl-tRNA_CAG gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNA_CAG gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNA^Asp, tRNA^Ala and tRNA^Ile. The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae . We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes.     

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux -marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. lux CDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn 4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of lux CDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 μg ml⁻¹, bioluminescence was stimulated (e.g. 218% of control), but at concentrations > 60 μg ml⁻¹ it declined to < 1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of ¹⁴CO₂ revealed that, for initial concentrations of 2.5–25 μg ml⁻¹, ≈55% of the added ¹⁴C was mineralized after 24 h compared with < 1% at 50 and 100 μg ml⁻¹. Inhibition of 2,4-DCP mineralization between 25 and 50 μg ml⁻¹ corresponded well to the EC₅₀ value (33.83 μg ml⁻¹) obtained from bioluminescence inhibition studies. lux -marked RASC c2 may therefore be used as a functionally (i.e. 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-σ28 domain

The flagellar-specific anti-sigma factor, FlgM, inhibits the expression of late flagellar genes until the hook–basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FlgM monitors this assembly checkpoint by being a substrate for export via the hook–basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FlgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FlgM. Deletion of this region (S3-P11) resulted in lower intracellular FlgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FlgM spanning residues K27 to A65 that exhibited increased anti-σ²⁸ activity. These FlgM 'hyperinhibitor' mutants were secreted less than wild-type FlgM. Mutations that interfere with the secretion of FlgM without abolishing anti-σ²⁸ activity have a negative effect upon the secretion of a His-tagged FlgM mutant that lacks anti-σ²⁸ activity. Models are proposed to explain the dominant negative phenotype of the FlgM secretion mutants reported in this study.     

Association of the 54-nucleotide repeat polymorphism of hPer3 with heroin dependence in Han Chinese population

Circadian clock genes have the function of producing circadian rhythm. They are also implicated in the origin or development of many diseases such as cancers and neuropsychiatric diseases. The purpose of this study is to determine whether the 54-nucleotide repeat polymorphism of hPer3 , one of the circadian clock genes, associates with heroin dependence. DNA samples were obtained from 209 Chinese heroin-dependent subjects and 249 Chinese healthy controls. The 54-nucleotide repeat polymorphism was detected by polymerase chain reaction and DNA agarose gel electrophoresis. The frequency of four-repeat allele was significantly higher ( χ ²= 10.64, P = 0.001; corrected for multiple tests, P = 0.003) in the mixed gender heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the mixed gender control group (four repeat: 0.81, five repeat: 0.19); the frequency of four-repeat allele was also significantly higher ( χ ²= 10.00, P = 0.002; corrected for multiple tests, P = 0.006) in the male heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the male control group (four repeat: 0.81, five repeat: 0.19); for females, no significant trend was observed with the 54-nucleotide repeat polymorphism between the heroin-dependent subject group and the control group. Our results suggest that the 54-nucleotide repeat polymorphism of hPer3 significantly associates with heroin dependence at the allele frequency level and may be a potential risk factor for the development of heroin dependence.     

Molecular Characterization of a Genomic Fragment Containing Pi-kh Gene from the Genomic Library of Indica Rice Line Tetep

Identification of full length genes along with upstream regulatory elements is important to understand its expression. Here, we report preparation of high titre genomic library and identification of a genomic clone containing Pi-k ^h gene with its complete upstream and downstream sequences from the rice blast resistant line Tetep. Structural analysis of protein revealed that Pi-k ^h has a central nucleotide binding site domain, leucine-rich repeats domain and a unique zinc-finger domain. Comparative analysis of Pi-k ^h protein sequence showed 64% and 45% similarity with the protein sequences of rice blast resistance genes Pi-b and Pi-ta , respectively.     

A genomic island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth

A 47 kb genomic island (GEI) bracketed by 50 bp direct repeats, containing 52 annotated genes, was found to delete spontaneously from the genome of Desulfovibrio vulgaris Hildenborough. The island contains genes for site-specific recombinases and transposases, rubredoxin:oxygen oxidoreductase-1 (Roo1) and hybrid cluster protein-1 (Hcp1), which promote survival in air and nitrite stress. The numbering distinguishes these from the Roo2 and Hcp2 homologues for which the genes are located elsewhere in the genome. Cells with and without the island (GEI⁺ and GEI^- cells respectively) were obtained by colony purification. GEI^- cells arise in anaerobic cultures of colony-purified GEI⁺ cells, indicating that the site-specific recombinases encoded by the island actively delete this region. GEI⁺ cells survive better in microaerophilic conditions due to the presence of Roo1, whereas the Hcps appear to prevent inhibition by sulfur and polysulfide, which are formed by chemical reaction of sulfide and nitrite. Hence, the island confers resistance to oxygen and nitrite stress. However, GEI^- cells have a higher growth rate in anaerobic media. Microarrays and enzyme activity stains indicated that the GEI^- cells have increased expression of genes, which promote anaerobic energy conservation, explaining the higher growth rate. Hence, while lowering the efficiency of anaerobic metabolism, the GEI increases the fitness of D. vulgaris under stress conditions, a feature reminiscent of pathogenicity islands which allow more effective colonization of environments provided by the targeted hosts.     

Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an 'extended −10' motif and a core −10 element

σ²⁸ controls the expression of flagella-related genes and is the most widely distributed alternative σ factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of σ²⁸ promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ²⁸ participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ²⁸. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ²⁸ orthologues. σ²⁸ is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.