The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

Caracterização eletretroforetica e expressão das fimbrias Fy e 31A de amostrasde Escherichia coli de origem bovina

Orientação: Wanderley Dias da SilveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Amostras de Escherichia coli portadoras das fímbrias FY e 31A, associadas a diarréia e septicemia em bovinos, foram estudadas visando determinar a localização da expressão gênica destas fímbrias. As amostras bacterianas 31A+: BZ43, BZ2468 e 31A e FY+: Att25, 54-5, 2147-4 e 11a, foram analisadas quanto à produção de enterotoxinas (STI e LT), VT, hemolisina, resistência a antibióticos, hemaglutinação D-manose resistente com eritrócitos e reação com antissoros específicos. A caracterização das fímbrias envolveu microscopia eletrônica, análise eletroforética de proteínas totais, de superfície e de membrana em SDS-PAGE e "Western Blotting". Foi também verificada a presença de plasmídios conjugativos nas amostras bacterianas e a caracterização dos transconjugantes para a expressão de FY e 31A. Uma amostra portadora de fímbria 31A (amostra BZ43) foi mutagenizada com transposon TnphoA. A expressão de 31A foi avaliada através de aglutinação com antissoros específicos, hemaglutinação de eritrócitos de boi, adesão a células Hela em cultura de tecido e patogenicidade em teste de competição em camundongo. A análise dos padrões de hemaglutinação, proteínas totais, proteínas de superfície e aglutinação com antissoros específicos permitiu a determinação das subunidades protéicas destas fímbrias como sendo aproximadamente 20 kDa para FY e 17 kDa para 31A. A amostra 11a, anteriormente classificada como portadora de fímbria FY, foi reclassificada como portadora de fímbria 31A através dos resultados de SDS-PAGE de proteínas de superfície e reação com antissoros específicos. As amostras Att25, BZ2468 e 31A apresentaram plasmídios conjugativos com marcas de resistência a antibióticos, porém não codificaram para a expressão de fímbrias nas linhagens transconjugantes, sugerindo que a expressão destas fímbrias ocorre a nível cromossômico. A amostra BZ43 mutagenizada com TnphoA originou duas amostras não-hemaglutinantes (eritrócitos bovinos) que apresentaram reação com o anticorpo específico anti-31A. A perda da capacidade de hemaglutinação foi relacionada à ausência da subunidade da fímbria 31A (17 kDa) em SDS-PAGE de proteínas de superfície. Uma das amostras mutantes apresentou fímbrias em microscopia eletrônica. A ausência defímbrias no outro mutante foi relacionada à não expressão de uma proteína de membrana de 35 kDa. A perda da capacidade de eritrócitos de bovino, associada ou não aglutinação de à expressão de fímbrias, não alterou o padrão de adesão a células HeLa dos mutantes (AD). A amostra BZ43 e o mutante fimbriado não apresentaram patogenicidade no teste de competição em camundongos de 3 dias de idade. A perda da capacidade de hemaglutinação parece não conferir uma menor adesão da amostra mutante nestes animaisAbstract: Escherichia coli strains associated with diarrhea and septicemic infections of cattle were studied to determine the genetic expression of FY and 31A fimbriae. The strains 31A+: BZ43, BZ2468 and 31A, and FY+: Att25, 54-5, 2147-4 and 11a, were evaluated for enterotoxin production (STI and LT), VT, hemolysin, antibiotic resistance, mannose resistant RBC hemagglutination, and reaction with specific antisera. The characterization of fimbriae involved electron microscopy, SDS-PAGE of total, surface and membrane proteins and Western blotting. Conjugative plasmids in the strains were transferred and analyzed for FY and 31A expression. Hemagglutination, SDS-PAGE of total and surface proteins and reaction with specific antisera allowed the location of the fimbrial subunits at 20 kDa for FY and 17 kDa for 31A. Strain 11a, originally grouped as FY by others, was classified as 31A based on SDS-PAGE of surface proteins and reaction with specific antisera. Strains Att25, BZ2468 and 31A had conjugative plasmids coding antibiotic resistance marks, but transconjugants failed to express the fimbriae, suggesting it could be located in the bacterial chromosome. Strain BZ43 (31A) was mutagenized using TnphoA transposon and mutants with negative hemagglutination of bovine RBC were assayed with specific antiserum, and tested for adhesion to HeLa cells in tissue culture and pathogenicity in the mouse competition assay. Loss of hemagglutinating capacity was related to the lack of the 31A fimbriae subunit (17 kDa) on SDS-PAGE of surface proteins. One mutant strain showed fimbriae under electron microscopy. The absence of fimbriae in the other mutant strain was related to a missing 35 kDa membrane protein. Both mutants reacted positively with absorbed anti-31A antiserum suggesting that fímbrial epithopes were expressed. Loss of bovine RBC hemagglutination did not altered the adhesion patter to HeLa cells (AD), either in the fimbriated or in the non-fimbriated mutant strain. Strain BZ43 and the fimbriated mutant strain were non-pathogenic to 3 day old mice in the competition assay. The lack of hemagglutination seemed not to interfere with adhesion properties of the mutant strain in this assayMestradoGeneticaMestre em Ciências Biológica

Genetic Variability And Pathogenicity Potential Of Escherichia Coli Isolated From Recreational Water Reservoirs.

Contamination of recreational waters and public water supplies by Escherichia coli represents a risk for public health, since some strains can be pathogenic or propagated with other pathogenic microorganisms. In this study, two reservoirs, Billings and Guarapiranga (São Paulo metropolitan area, Brazil), were investigated in order to assess E. coli diversity. Genetic typing using rep-PCR completely differentiated all strains and enabled the determination of their genetic variability. Although the same level of genetic variability was observed for strains originating from both reservoirs, randomization procedures showed that isolates from the same reservoir were more closely related to each other. Phylogenetic group frequencies in each reservoir suggested that contamination in the Billings reservoir was mostly from humans, whereas contamination in the Guarapiranga reservoir was mostly from animals. Colony blot experiments using probes from several virulence factor genes showed that both reservoirs contained potential pathogenic strains and may represent a risk to recreational or household usage of these water resources.158420-

Differentiation Of Acidithiobacillus Ferrooxidans And A. Thiooxidans Strains Based On 16s-23s Rdna Spacer Polymorphism Analysis.

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.155559-6

Characterization of selected strains of mucorales using fatty acid profiles Caracterização de linhagens de mucorales através do perfil de ácidos graxos

The fatty acid profiles of several fungi of the order Mucorales (Zygomycetes), including Backusella lamprospora (Lendner) Benny and R.K. Benj., Benjaminiella youngii P.M. Kirk, Circinella simplex van Tieghem, Cunninghamella blakesleeana Lendner, Mortierella ramanniana (Möller) Linnem., Mucor circinelloides f. janssenii (Lendner) Schipper, Mycotypha microspora Fenner, Rhizomucor miehei (Cooney and R. Emerson) Schipper and Rhizomucor pusillus (Lindt) Schipper, and of Volutella sp. Fr., from the class Ascomycetes, were qualitatively analysed by gas-liquid chromatography in order to determine the taxonomic value of these chemotaxonomic markers. The fatty acids present in all strains were palmitic (16:0), oleic (18:1), linoleic (18:2) and <FONT FACE="Symbol">g</FONT>-linolenic (18:3) acid, with the exception that the latter was not found in Volutella sp. Chemotaxonomic markers for some species and genera were obtained, including a non-identified fatty acid, FAME8 (minimum and maximum retention times of 27.92 and 28.28 minutes) for Rhizomucor miehei CCT 2236 and Rhizomucor pusillus CCT 4133, and FAME3 (minimum and maximum of 16.53 and 16.61 minutes) for Benjaminiella youngii CCT 4121. The chemotaxonomic marker of the order Mucorales was the fatty acid 18:3<FONT FACE="Symbol">w</FONT>6, confirming previous data from literature. The results of the present study suggest that qualitative fatty acid analysis can be an important chemotaxonomic tool for the classification of fungi assigned to the order Mucorales (Zygomycetes).<br>O perfil de ácidos graxos de Backusella lamprospora (Lendner) Benny e R.K. Benj., Benjaminiella youngii P.M. Kirk, Circinella simplex van Tieghem, Cunninghamella blakesleeana Lendner, Mortierella ramanniana (Möller) Linnem., Mucor circinelloides f. janssenii (Lendner) Schipper, Mycotypha microspora Fenner, Rhizomucor miehei (Cooney e R. Emerson) Schipper e Rhizomucor pusillus (Lindt) Schipper, da ordem Mucorales (Zygomycetes), e Volutella sp. Fr., da classe Ascomycetes, foram analisados qualitativamente por cromatografia gás-líquida, tendo como objetivo determinar o valor taxonômico destes marcadores quimiotaxonômicos. Os ácidos palmítico (16:0), oléico (18:1), linoléico (18:2) e <FONT FACE="Symbol">g</FONT>-linolênico (18:3) foram encontrados em todas as linhagens, com exceção do último, o qual não foi encontrado na linhagem de Volutella analisada. Foram obtidos marcadores quimiotaxonômicos para algumas espécies e gêneros estudados, incluindo um ácido graxo não-identificado, FAME8 (tempos de retenção mínimo e máximo de 27,92 e 28,28 minutos) para Rhizomucor miehei CCT 2236 e Rhizomucor pusillus CCT 4133 e FAME3 (tempos de retenção mínimo e máximo de 16,53 e 16,61 minutos) para Benjaminiella youngii CCT 4121. Para a ordem Mucorales, o marcador quimiotaxonômico obtido foi o ácido graxo 18:3<FONT FACE="Symbol">w</FONT>6, confirmando dados da literatura. Os resultados do presente estudo sugerem que a análise qualitativa do perfil de ácidos graxos pode ser uma ferramenta importante na classificação de fungos da ordem Mucorales (Zygomycetes)

Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae