Freezing and thawing in Antarctica: characterization of antifreeze protein (AFP) producing microorganisms isolated from King George Island, Antarctica.

Antarctic temperature variations and long periods of freezing shaped the evolution of microorganisms with unique survival mechanisms. These resilient organisms exhibit several adaptations for life in extreme cold. In such ecosystems, microorganisms endure the absence of liquid water and exhibit resistance to freezing by producing water-binding molecules such as antifreeze proteins (AFP). AFPs modify the ice structure, lower the freezing point, and inhibit recrystallization. The objective of this study was to select and identify microorganisms isolated from different Antarctic ecosystems based on their resistance to temperatures below 0 °C. Furthermore, the study sought to characterize these microorganisms regarding their potential antifreeze adaptive mechanisms. Samples of soil, moss, permafrost, and marine sediment were collected on King George Island, located in the South Shetland archipelago, Antarctica. Bacteria and yeasts were isolated and subjected to freezing-resistance and ice recrystallization inhibition (IR) tests. A total of 215 microorganisms were isolated, out of which 118 were molecularly identified through molecular analysis using the 16S rRNA and ITS regions. Furthermore, our study identified 24 freezing-resistant isolates, including two yeasts and 22 bacteria. A total of 131 protein extracts were subjected to the IR test, revealing 14 isolates positive for AFP production. Finally, four isolates showed both freeze-resistance and IR activity (Arthrobacter sp. BGS04, Pseudomonas sp. BGS05, Cryobacterium sp. P64, and Acinetobacter sp. M1_25C). This study emphasizes the diversity of Antarctic microorganisms with the ability to tolerate freezing conditions. These microorganisms warrant further investigation to conduct a comprehensive analysis of their antifreeze capabilities, with the goal of exploring their potential for future biotechnological applications.

Diversity of hydrocarbon-degrading Klebsiella strains isolated from hydrocarbon-contaminated estuaries.

AIMS: To investigate the diversity and the catabolic capacity of oil-degrading Klebsiella strains isolated from hydrocarbon-contaminated sediments in Santos-São Vicente estuary systems in Brazil. METHODS AND RESULTS: Klebsiella strains obtained from the estuary were characterized using 16S rRNA gene sequencing and BOX-PCR patterns, testing their catabolic capacity to degrade toluene, xylene, naphthalene and nonane, and identifying the catabolic genes present in the oil-degrading strains. Results show that Klebsiella strains were widespread in the estuary. Twenty-one isolates from the Klebsiella genus were obtained; 14 had unique BOX patterns and were further investigated. Among four distinct catabolic genes tested (todC1, ndoB, xylE and alkB1), only the todC1 gene could be amplified in two Klebsiella strains. The biodegradation assay showed that most of the strains had the ability to degrade all of the tested hydrocarbons; however, the strains displayed different efficiencies. CONCLUSIONS: The oil-degrading Klebsiella isolates obtained from the estuary were closely related to Klebsiella pneumoniae and Klebsiella ornithinolytica. The isolates demonstrated a substantial degree of catabolic plasticity for hydrocarbon degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that several strains from the Klebsiella genus are able to degrade diverse hydrocarbon compounds. These findings indicate that Klebsiella spp. can be an important part of the oil-degrading microbial community in estuarine areas exposed to sewage.

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil.

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.

Evaluation of strains isolated by growth on naphthalene and biphenyl for hybridization of genes to dioxygenase probes and polychlorinated biphenyl-degrading ability.

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.

Assessment of media using B-D-Glucoronidase activity for the detection of Escherichia coli in water

Quatro meios de cultura contendo B-D-glucoronide MUG (Lauril Sulfato Fluorocult, ECD Fluorocult Agar, Caldo Lauril Triptose e Agar nutriente suplementados com MUG) foram comparados com meios convencionais (caldo Lauril Triptose, Caldo E.C. e Agar M-Endo LES) para verificar se havia diferenças entre eles na detecçäo de E. coli. As amostras de água foram contaminadas artificialmente com E.coli isoladas tanto do ambiente quanto de espécimes clínicos e analisados pelas técnicas dos tubos múltiplos e de membrana filtrante usando os meios acima descritos. Entre as cepas previamente analisadas para o preparo das amostras artificialmente contaminadas, 6,4 por cento näo apresentaram atividade B-D-Glucoronidase quando testadas com meio contendo MUG. Os resultados obtidos foram submetidos ao Teste de Wilcoxon que mostrou que Lauril Sulfato Fluorocult, caldo Lauril Triptose com MUG e Agar Nutriente com MUG foram täo sensíveis quanto o Caldo Lauril Triptose e meio E.C. e Agar M-Endo LES, e que o ECD Fluorocult Agar apresentou uma sensibilidade muito baixa. Esta técnica é de grande importância pois pode ser aplicada em métodos mais rápidos para detecçäo de E.coli e em automaçäo

[Bacteriological quality of groundwaters in cemeteries]. / Qualidade bacteriológica de águas subterrâneas em cemitérios.

Groundwater samples collected by piezometers from three cemeteries in geologically distinct areas of S. Paulo and Santos, Brazil, were analysed in order to determine their hygienic and sanitary conditions. Fecal coliformes, fecal streptococci, sulfite reducer clostridia and Salmonella were searched for the purpose of evaluating sanitary conditions, and total coliforms, heterotrophic bacteria, proteolitic and lipolitic microorganisms for evaluating hygienic conditions. In some samples, nitrate levels were also determined. It was discovered that these waters do not present adequate sanitary and hygienic conditions and that, in some cases, nitrate levels were extremely high (75.7 mg/l). In most samples, higher levels of fecal streptococci and sufite reducer clostridia than fecal coliforms were detected, which seems to show that the two former indicators would be more appropriate for evaluating the sanitary conditions of this kind of water. Salmonella were detected in only one of 44 samples analysed and coliphages in none. In the statistical analysis, the correlation matrix showed significant correlations among three fecal pollution indicators, as well as among anaerobic and aerobic heterotrophs and lipolitic bacteria. A direct relationship between the deterioration of water quality and the geological and hydrogeological conditions of the environment studied was observed. When cemeteries are constructed these conditions should, therefore, be taken into consideration.