Sea-Ice Bacteria Halomonas sp. Strain 363 and Paracoccus sp. Strain 392 Produce Multiple Types of Poly-3-Hydroxyalkaonoic Acid (PHA) Storage Polymers at Low Temperature.

Poly-3-hydroxyalkanoic acids (PHAs) are bacterial storage polymers commonly used in bioplastic production. Halophilic bacteria are industrially interesting organisms, as their salinity tolerance and psychrophilic nature lowers sterility requirements and subsequent production costs. We investigated PHA synthesis in two bacterial strains, Halomonas sp. 363 and Paracoccus sp. 392, isolated from Southern Ocean sea ice and elucidated the related PHA biopolymer accumulation and composition with various approaches, such as transcriptomics, microscopy, and chromatography. We show that both bacterial strains produce PHAs at 4°C when the availability of nitrogen and/or oxygen limited growth. The genome of Halomonas sp. 363 carries three phaC synthase genes and transcribes genes along three PHA pathways (I to III), whereas Paracoccus sp. 392 carries only one phaC gene and transcribes genes along one pathway (I). Thus, Halomonas sp. 363 has a versatile repertoire of phaC genes and pathways enabling production of both short- and medium-chain-length PHA products. IMPORTANCE Plastic pollution is one of the most topical threats to the health of the oceans and seas. One recognized way to alleviate the problem is to use degradable bioplastic materials in high-risk applications. PHA is a promising bioplastic material as it is nontoxic and fully produced and degraded by bacteria. Sea ice is an interesting environment for prospecting novel PHA-producing organisms, since traits advantageous to lower production costs, such as tolerance for high salinities and low temperatures, are common. We show that two sea-ice bacteria, Halomonas sp. 363 and Paracoccus sp. 392, are able to produce various types of PHA from inexpensive carbon sources. Halomonas sp. 363 is an especially interesting PHA-producing organism, since it has three different synthesis pathways to produce both short- and medium-chain-length PHAs.

Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms.

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all <96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity<70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed.

Association between dyslipidemia and anthropometric indicators in adolescents.

The dyslipidemia associated with excess weight is a risk profile global call for cardiovascular disease (CVD). The aim of this study was to investigate the association between dyslipidemias and other risk factors for cardiovascular diseases (CVD) in adolescents, considering sexual maturation. A cross-sectional study was carried out with 432 adolescents from public schools, aged 10-19 years. The correlations between the variables from the lipid profile and the Body Mass Index (BMI), waist circumference (WC), waist-to-height ratio (WHtR), sexual maturation, familial history and maternal education were evaluated using Pearson's correlation coefficient. Low high-density lipoprotein cholesterol (HDL-C) was the most prevalent dyslipidemia (50.5%), regardless of gender. There were significant correlations between triglycerides and BMI (r = 0.30, p<0.01), WC (r = 0.32, p < 0.01) and WHtR (r = 0.33, p < 0.01). The linear model, which took into consideration sexual maturation, age and BMI, explain about 1 to 10.4% of the lipid profile variation. The low HDL-c was the most prevalent dyslipidemia in all adolescents and hypertriglyceridemia was most prevalent in overweight adolescents. Associations between dyslipidemias and anthropometric indicators (BMI and RCA) detected in this study can generate the hypothesis of the risk factors for CVD in adolescents.

Association between dyslipidemia and anthropometric indicators in adolescents / Asociación entre la dislipemia y los indicadores antropometricos en adolescentes

The dyslipidemia associated with excess weight is a riskprofile global call for cardiovascular disease (CVD). The aim of this study was to investigate the association between dyslipidemias and other risk factors for cardiovascular diseases (CVD) in adolescents, considering sexual maturation. A cross-sectional study was carried outwith 432 adolescents from public schools, aged 10-19years. The correlations between the variables from the lipid profile and the Body Mass Index (BMI), waist circumference(WC), waist-to-height ratio (WHtR), sexual maturation, familial history and maternal education were evaluated using Pearson’s correlation coefficient. Low high-density lipoprotein cholesterol (HDL-C) was the most prevalent dyslipidemia (50.5%), regardless ofgender. There were significant correlations between triglycerides and BMI (r = 0.30, p<0.01), WC (r = 0.32, p <0.01) and WHtR (r = 0.33, p < 0.01). The linear model,which took into consideration sexual maturation, age and BMI, explain about 1 to 10.4% of the lipid profile variation. The low HDL-c was the most prevalent dyslipidemia in all adolescents and hypertriglyceridemia was most prevalent in overweight adolescents. Associations between dyslipidemias and anthropometric indicators (BMI andRCA) detected in this study can generate the hypothesis of the risk factors for CVD in adolescents (AU)

La dislipidemia asociada con el exceso de peso es un perfil de riesgo de alcance mundial para la enfermedad cardiovascular (ECV). El objetivo de este estudio fue investigar la asociación entre la dislipemia y otros factores de riesgo para enfermedad cardiovascular (ECV) en los adolescentes en edad púber. Se realizó un estudio transversal con 432 adolescentes de escuelas públicas, con edades entre 10-19 años. Estudiando las correlaciones entre las variables del perfil lipídico y el índice de masa corporal (IMC), la circunferencia de cintura (CC), la cintura/altura (RCA) y la maduración sexual. Los antecedentes familiares y la educación de la madre se evaluaron mediante el coeficiente de correlación de Pearson. La dislipidemia com bajos niveles de HDL-C fue más frecuente (50,5%), independientemente del género. Se observaron correlaciones significativas entre los triglicéridos y el IMC (r = 0,30, p < 0,01), CC (r = 0,32, p < 0,01) y RCA (r = 0,33, p < 0, 01). El modelo lineal, teniendo en cuenta la maduración sexual, la edad, y el IMC, explicó entre el 1 y el 10,4% de la variación del perfil lipídico. Los bajos niveles de HDL-C fueron la dislipidemia más frecuente en todos los adolescentes y la hipertrigliceridemia en adolescentes con sobrepeso. Las asociaciones entre las dislipidemias y los indicadores antropométricos (índice de masa corporal y RCA) detectado en este estudio, pueden generar hipótesis acerca de los factores de riesgo de ECV en los adolescentes (AU)

Hearing evaluation in newborns with congenital aural malformation.

OBJECTIVES: Over 5 years, 68,472 newborns were screened through a newly implemented universal newborn hearing screening program. In 15 cases, atresia of the external ear canal was found. The aim of this study was to estimate the hearing status of these newborns using transiently evoked otoacoustic emissions (TEOAEs) and auditory brainstem responses (ABRs). METHODS: TEOAEs were performed during the first days after birth in the normal ears of all newborns. Diagnostic ABR audiometry was performed in 10 newborns. RESULTS: Unilateral involvement occurred in all newborns studied. TEOAEs were present in all the contralateral ears. Normal ABRs were recorded from the healthy ear, whereas a conductive hearing loss, of approximately 50-60 dB, was found in the involved ear. In long-term follow-up, a satisfactory level of hearing, language, and speech development was found in 9 of the newborns; it was too early to come to definite conclusions in 4 newborns and 2 newborns were lost in follow-up. CONCLUSIONS: In this study, all newborns with aural atresia had normal function in the contralateral ear. Yet, a comprehensive assessment of hearing is essential as early intervention is necessary in the rare case of bilateral hearing impairment.

Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera.

Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing. With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins. The respective sequence similarities were 99.9-100%, 94.2-99.9% and 99.3-100%. Thus the morphological characteristics (e.g. Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied. In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR. All the strains except two identical pairs of Microcystis strains had different band profiles. The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar. Based on the 16S rRNA gene sequence analysis, four major clades were formed. (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains. The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains.

Site-specific restriction endonucleases in cyanobacteria.

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.

Characterization of Nodularia strains, cyanobacteria from brackish waters, by genotypic and phenotypic methods.

An investigation was undertaken of the genetic diversity of Nodularia strains from the Baltic Sea and from Australian waters, together with the proposed type strain of Nodularia spumigena. The Nodularia strains were characterized by using a polyphasic approach, including RFLP of PCR-amplified 16S rRNA genes, 16S rRNA gene sequencing, Southern blotting of total DNA, repetitive extragenic palindromic- and enterobacterial repetitive intergenic consensus-PCR, ribotyping and phenotypic tests. With genotypic methods, the Nodularia strains were grouped into two clusters. The genetic groupings were supported by one phenotypic property: the ability to produce nodularin. In contrast, the cell sizes of the strains were not different in the two genetic clusters. 16S rRNA gene sequences indicated that all the Nodularia strains were closely related, despite their different origins. According to this study, two genotypes of Nodularia exist in the Baltic Sea. On the basis of the taxonomic definitions of Komarek et al. (Algol Stud 68, 1-25, 1993), the non-toxic type without gas vesicles fits the description of Nodularia sphaerocarpa, whereas the toxic type with gas vesicles resembles the species N. spumigena and Nodularia baltica.

Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene.

Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera.

Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli.

Cyanobacterial hepatotoxins, microcystins, are specific inhibitors of serine/threonine protein phosphatases and potent tumor promoters. They have caused several poisonings of animals and also pose a health hazard for humans through the use of water for drinking and recreation. Different strains of the same cyanobacterial species may variously be nontoxic, be neurotoxic, or produce several microcystin variants. It is poorly understood how the amount of toxins varies in a single strain. This laboratory study shows the importance of external growth stimuli in regulating the levels and relative proportions of different microcystin variants in two strains of filamentous, nitrogen-fixing Anabaena spp. The concentration of the toxins in the cells increased with phosphorus. High temperatures (25 to 30 degrees C), together with the highest levels of light studied (test range, 2 to 100 mumol m-2 s-1), decreased their amount. Different structural variants of microcystins responded differently to growth stimuli. Variants of microcystin (MCYST)-LR correlated with temperatures below 25 degrees C, and those of MCYST-RR correlated with higher temperatures. Nitrogen added into the growth medium and increasing temperatures increased the proportion of microcystin variants demethylated in amino acid 3. All variants remained mostly intracellular. Time was the most important factor causing the release of the toxins into the growth medium. Time, nitrogen added into the growth medium, and light fluxes above 25 mumol m-2 s-1 significantly increased the concentrations of the dissolved toxins. According to the results, it thus seems that the reduction of phosphorus loads in bodies of water might play a role in preventing the health hazards that toxic cyanobacterial water blooms pose, not only by decreasing the cyanobacteria but also by decreasing their toxin content.

High-frequency transfer of linear DNA containing 5'-covalently linked terminal proteins: electroporation of bacteriophage PRD1 genome into Escherichia coli.

Using electroporation with the phage PRD1 genome, we set up a high-frequency DNA transfer system for a linear dsDNA molecule with 5'-covalently linked terminal proteins. The transfer was saturated when more than 100 ng of PRD1 genome was used. Electroporation efficiency was about four orders of magnitude higher than that obtained with transfection. Removal of the terminal protein abolished plaque formation, which could not be rescued by supplying the terminal protein or phage DNA polymerase or both in trans.