Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.     

Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C·t value (product of the disinfectant concentration and contact time) of 60 mg·min·liter⁻¹, frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C·t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4°C to 25°C) and a decrease in pH result in better inactivation.     

Meta-Analysis of Genome-Wide Scans for Human Adult Stature Identifies Novel Loci and Associations with Measures of Skeletal Frame Size

Recent genome-wide (GW) scans have identified several independent loci affecting human stature, but their contribution through the different skeletal components of height is still poorly understood. We carried out a genome-wide scan in 12,611 participants, followed by replication in an additional 7,187 individuals, and identified 17 genomic regions with GW-significant association with height. Of these, two are entirely novel (rs11809207 in CATSPER4, combined P-value=6.1×10⁻⁸ and rs910316 in TMED10, P-value=1.4×10⁻⁷) and two had previously been described with weak statistical support (rs10472828 in NPR3, P-value=3×10⁻⁷ and rs849141 in JAZF1, P-value=3.2×10⁻¹¹). One locus (rs1182188 at GNA12) identifies the first height eQTL. We also assessed the contribution of height loci to the upper- (trunk) and lower-body (hip axis and femur) skeletal components of height. We find evidence for several loci associated with trunk length (including rs6570507 in GPR126, P-value=4×10⁻⁵ and rs6817306 in LCORL, P-value=4×10⁻⁴), hip axis length (including rs6830062 at LCORL, P-value=4.8×10⁻⁴ and rs4911494 at UQCC, P-value=1.9×10⁻⁴), and femur length (including rs710841 at PRKG2, P-value=2.4×10⁻⁵ and rs10946808 at HIST1H1D, P-value=6.4×10⁻⁶). Finally, we used conditional analyses to explore a possible differential contribution of the height loci to these different skeletal size measurements. In addition to validating four novel loci controlling adult stature, our study represents the first effort to assess the contribution of genetic loci to three skeletal components of height. Further statistical tests in larger numbers of individuals will be required to verify if the height loci affect height preferentially through these subcomponents of height.     

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Meta-Analysis of 28,141 Individuals Identifies Common Variants within Five New Loci That Influence Uric Acid Concentrations

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p=5.2×10⁻²⁰¹), ABCG2 (p=3.1×10⁻²⁶), SLC17A1 (p=3.0×10⁻¹⁴), SLC22A11 (p=6.7×10⁻¹⁴), SLC22A12 (p=2.0×10⁻⁹), SLC16A9 (p=1.1×10⁻⁸), GCKR (p=1.4×10⁻⁹), LRRC16A (p=8.5×10⁻⁹), and near PDZK1 (p=2.7×10⁻⁹). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p=4.0×10⁻²⁶) and propionyl-L-carnitine (p=5.0×10⁻⁸) concentrations, which in turn were associated with serum UA levels (p=1.4×10⁻⁵⁷ and p=8.1×10⁻⁵⁴, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.     

Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths of Escherichia coli KO11 by Expression of Pseudomonas putida estZ Esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter⁻¹. Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. The K_m and V_max for α-naphthyl acetate were 18 μM and 48.1 μmol·min⁻¹·mg of protein⁻¹, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol·min⁻¹·mg of protein⁻¹), followed by ethyl acetate (66 μmol·min⁻¹·mg of protein⁻¹). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter⁻¹.     

NRXN3 Is a Novel Locus for Waist Circumference: A Genome-Wide Association Study from the CHARGE Consortium

Central abdominal fat is a strong risk factor for diabetes and cardiovascular disease. To identify common variants influencing central abdominal fat, we conducted a two-stage genome-wide association analysis for waist circumference (WC). In total, three loci reached genome-wide significance. In stage 1, 31,373 individuals of Caucasian descent from eight cohort studies confirmed the role of FTO and MC4R and identified one novel locus associated with WC in the neurexin 3 gene [NRXN3 (rs10146997, p=6.4×10⁻⁷)]. The association with NRXN3 was confirmed in stage 2 by combining stage 1 results with those from 38,641 participants in the GIANT consortium (p=0.009 in GIANT only, p=5.3×10⁻⁸ for combined analysis, n=70,014). Mean WC increase per copy of the G allele was 0.0498 z-score units (0.65 cm). This SNP was also associated with body mass index (BMI) [p=7.4×10⁻⁶, 0.024 z-score units (0.10 kg/m²) per copy of the G allele] and the risk of obesity (odds ratio 1.13, 95% CI 1.07–1.19; p=3.2×10⁻⁵ per copy of the G allele). The NRXN3 gene has been previously implicated in addiction and reward behavior, lending further evidence that common forms of obesity may be a central nervous system-mediated disorder. Our findings establish that common variants in NRXN3 are associated with WC, BMI, and obesity.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.     

Genome-Wide Association Scan Meta-Analysis Identifies Three Loci Influencing Adiposity and Fat Distribution

To identify genetic loci influencing central obesity and fat distribution, we performed a meta-analysis of 16 genome-wide association studies (GWAS, N=38,580) informative for adult waist circumference (WC) and waist–hip ratio (WHR). We selected 26 SNPs for follow-up, for which the evidence of association with measures of central adiposity (WC and/or WHR) was strong and disproportionate to that for overall adiposity or height. Follow-up studies in a maximum of 70,689 individuals identified two loci strongly associated with measures of central adiposity; these map near TFAP2B (WC, P=1.9×10⁻¹¹) and MSRA (WC, P=8.9×10⁻⁹). A third locus, near LYPLAL1, was associated with WHR in women only (P=2.6×10⁻⁸). The variants near TFAP2B appear to influence central adiposity through an effect on overall obesity/fat-mass, whereas LYPLAL1 displays a strong female-only association with fat distribution. By focusing on anthropometric measures of central obesity and fat distribution, we have identified three loci implicated in the regulation of human adiposity.     

A genome-wide association study identifies novel alleles associated with hair color and skin pigmentation

We conducted a multi-stage genome-wide association study of natural hair color in more than 10,000 men and women of European ancestry from the United States and Australia. An initial analysis of 528,173 single nucleotide polymorphisms (SNPs) genotyped on 2,287 women identified IRF4 and SLC24A4 as loci highly associated with hair color, along with three other regions encompassing known pigmentation genes. We confirmed these associations in 7,028 individuals from three additional studies. Across these four studies, SLC24A4 rs12896399 and IRF4 rs12203592 showed strong associations with hair color, with p=6.0×10⁻⁶² and p=7.46×10⁻¹²⁷, respectively. The IRF4 SNP was also associated with skin color (p=6.2×10⁻¹⁴), eye color (p=6.1×10⁻¹³), and skin tanning response to sunlight (p=3.9×10⁻⁸⁹). A multivariable analysis pooling data from the initial GWAS and an additional 1,440 individuals suggested that the association between rs12203592 and hair color was independent of rs1540771, a SNP between the IRF4 and EXOC2 genes previously found to be associated with hair color. After adjustment for rs12203592, the association between rs1540771 and hair color was not significant (p=0.52). One variant in the MATP gene was associated with hair color. A variant in the HERC2 gene upstream of the OCA2 gene showed the strongest and independent association with hair color compared with other SNPs in this region, including three previously reported SNPs. The signals detected in a region around the MC1R gene were explained by MC1R red hair color alleles. Our results suggest that the IRF4 and SLC24A4 loci are associated with human hair color and skin pigmentation.     

Effect of turgor pressure and cell size on the wall elasticity of plant cells

Direct measurements of the volumetric elastic modulus, , of cells of a higher plant were performed on the epidermal bladder cells of Mesembryanthemum crystallinum using a pressure probe technique. Measurements on giant algal cells (Valonia, Nitellopsis) are given for comparison. Giant celled algae and M. crystallinum bladders have elastic moduli, , which depend strongly on turgor pressure, P, and on cell volume, V. The values of Mesembryanthemum bladders range between 5 bar at zero pressure and 100 bar at full turgor pressure (3-4 bar). increased with cell size (volume) at a given turgor pressure, and this volume dependence was pronounced more in the high pressure range. From the (P) characteristics, complete volume-pressure curves were obtained for Mesembryanthemum bladders and giant algal cells. The results suggest that the (P) and (V) characteristics of all plant cells are similar. The significance of the pressure and volume effects for the water relations and growth processes of plant cells is discussed briefly.     

Specificity of the STAT4 genetic association for severe disease manifestations of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n=1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r²=0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF)=31.1% in SLE cases compared with 22.5% in controls (OR=1.56, p=10⁻¹⁶). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF=35.1%, OR=1.86, p<10⁻¹⁹), nephritis (MAF=34.3%, OR=1.80, p<10⁻¹¹), and age at diagnosis<30 years (MAF=33.8%, OR=1.77, p<10⁻¹³). An association with severe nephritis was even more striking (MAF=39.2%, OR=2.35, p<10⁻⁴ in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.     

High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Characterization of Temperate Bacillus Bacteriophage phi105

Temperate Bacillus phage 105 is serologically unrelated to previously described virulent Bacillus phages. Phage 105 is incapable of generalized transduction. Prophage 105 is inducible with mitomycin C. Phage 105 contains double-stranded deoxyribonucleic acid (DNA) with a molecular weight of about 25 × 10⁷ as determined by band sedimentation and electron microscopy. The per cent guanine plus cytosine of 105 DNA is 43.5 as determined by buoyant density in CsCl and by thermal denaturation. Phage 105 DNA may contain complementary single-stranded ends.     

Genome-Wide Association Study of Plasma Polyunsaturated Fatty Acids in the InCHIANTI Study

Polyunsaturated fatty acids (PUFA) have a role in many physiological processes, including energy production, modulation of inflammation, and maintenance of cell membrane integrity. High plasma PUFA concentrations have been shown to have beneficial effects on cardiovascular disease and mortality. To identify genetic contributors of plasma PUFA concentrations, we conducted a genome-wide association study of plasma levels of six omega-3 and omega-6 fatty acids in 1,075 participants in the InCHIANTI study on aging. The strongest evidence for association was observed in a region of chromosome 11 that encodes three fatty acid desaturases (FADS1, FADS2, FADS3). The SNP with the most significant association was rs174537 near FADS1 in the analysis of arachidonic acid (AA; p=5.95×10⁻⁴⁶). Minor allele homozygotes had lower AA compared to the major allele homozygotes and rs174537 accounted for 18.6% of the additive variance in AA concentrations. This SNP was also associated with levels of eicosadienoic acid (EDA; p=6.78×10⁻⁹) and eicosapentanoic acid (EPA; p=1.07×10⁻¹⁴). Participants carrying the allele associated with higher AA, EDA, and EPA also had higher low-density lipoprotein (LDL-C) and total cholesterol levels. Outside the FADS gene cluster, the strongest region of association mapped to chromosome 6 in the region encoding an elongase of very long fatty acids 2 (ELOVL2). In this region, association was observed with EPA (rs953413; p=1.1×10⁻⁶). The effects of rs174537 were confirmed in an independent sample of 1,076 subjects participating in the GOLDN study. The ELOVL2 SNP was associated with docosapentanoic and DHA but not with EPA in GOLDN. These findings show that polymorphisms of genes encoding enzymes in the metabolism of PUFA contribute to plasma concentrations of fatty acids.     

Multiple Loci within the Major Histocompatibility Complex Confer Risk of Psoriasis

Psoriasis is a common inflammatory skin disease characterized by thickened scaly red plaques. Previously we have performed a genome-wide association study (GWAS) on psoriasis with 1,359 cases and 1,400 controls, which were genotyped for 447,249 SNPs. The most significant finding was for SNP rs12191877, which is in tight linkage disequilibrium with HLA-Cw*0602, the consensus risk allele for psoriasis. However, it is not known whether there are other psoriasis loci within the MHC in addition to HLA-C. In the present study, we searched for additional susceptibility loci within the human leukocyte antigen (HLA) region through in-depth analyses of the GWAS data; then, we followed up our findings in an independent Han Chinese 1,139 psoriasis cases and 1,132 controls. Using the phased CEPH dataset as a reference, we imputed the HLA-Cw*0602 in all samples with high accuracy. The association of the imputed HLA-Cw*0602 dosage with disease was much stronger than that of the most significantly associated SNP, rs12191877. Adjusting for HLA-Cw*0602, there were two remaining association signals: one demonstrated by rs2073048 (p=2×10⁻⁶, OR=0.66), located within c6orf10, a potential downstream effecter of TNF-alpha, and one indicated by rs13437088 (p=9×10⁻⁶, OR=1.3), located 30 kb centromeric of HLA-B and 16 kb telomeric of MICA. When HLA-Cw*0602, rs2073048, and rs13437088 were all included in a logistic regression model, each of them was significantly associated with disease (p=3×10⁻⁴⁷, 6×10⁻⁸, and 3×10⁻⁷, respectively). Both putative loci were also significantly associated in the Han Chinese samples after controlling for the imputed HLA-Cw*0602. A detailed analysis of HLA-B in both populations demonstrated that HLA-B*57 was associated with an increased risk of psoriasis and HLA-B*40 a decreased risk, independently of HLA-Cw*0602 and the C6orf10 locus, suggesting the potential pathogenic involvement of HLA-B. These results demonstrate that there are at least two additional loci within the MHC conferring risk of psoriasis.     

Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of Nɛ-Acetyl-β-Lysine and Growth at High Salinity

The compatible solute N-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze N-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced N-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.

     

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage 50, interferes with 50 replication. The per50 fragment was found to afford negligible protection to NCK203 against 50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected 50 infection, reducing the efficiency of plaquing (EOP) to 2.5 × 10^-4 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage 31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on 31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of 31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 × 10^-7 but the plaques were large and contained Per31-resistant phages. Characterization of these “new” phages revealed at least two different types that were similar to 31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.     

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T_m value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t_1/2 values of>24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t_1/2=1631min) compared with FRNA (t_1/2=53.2min) and MeRNA (t_1/2=187min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.     

Application of a Specific and Sensitive Radiometric Assay for Microbial Lipase Activities in Marine Water Samples from the Lagoon of Nouméa

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [³H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [³H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA·liter⁻¹·h⁻¹ and from 0.76 to 0.23 nmol of ³H-FFA·liter⁻¹·h⁻¹, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 μg of C·liter⁻¹·h⁻¹). However, the ratio of MUF-oleate activities to [³H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.