Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Draft Genome Sequence of the Nitrogen-Fixing Symbiotic Bacterium Bradyrhizobium elkanii 587

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

STUDY OF AMNONIFYING BACTERIA BEHAVIOR IN THE LAKES CARIOCA AND D-HELVECIO (RIO-DOCE-VALLEY-MG)

The study of the amnonifying bacteria behavior in the lakes Carioca and D. Helvecio, which belong to the natural lacustrine system of the Rio Doce Valley - Minas Gerais - Brazil, during the period from March to November, showed a similar seasonal variation in both lakes, with higher population density in March and November (raining period) and lower density in May, July and September (dry period). The vertical variation was also relatively similar for both lakes, were during the thermal stratification the distribution was regular along the water column, while during the period of thermal stratification the populational density was remarkably different at different depths. The characterized ammonifying types to the genera Acinetobacter, Moraxella and Proteus, which in culture reveal high amnonifying activity with values which have reached 30,8 mug/l of ammonia per population unity (105 bacteria/ml).

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

A bacteriological study of hospital beds before and after disinfection with phenolic disinfectant

In hospitals, one of the ways to control microbial contamination is by disinfecting the furniture used by patients. This study's main objective was to evaluate the microbiological condition of hospital mattresses before and after such disinfection, in order to identify bacteria that are epidemiologically important in nosocomial infection, such as Staphylococcus aureus and Pseudomonas aeruginosa. RODAC plates with two different culture media were used to collect specimens. Patient beds were selected according to previously established criteria, and surface areas on the mattresses were chosen at random. From the total of 1 040 plate cultures from 52 mattresses, positive results were obtained from 500 of them (48.1%), 263 before disinfection and 237 after disinfection. Considering the selectivity of the culture media, the positivity rate was high. There were high prevalences of S. aureus both before and after mattress disinfection. The study results suggest that the usual disinfection procedures, instead of diminishing the number of microbes, merely displace them from one part of the mattress to another, and the number of microorganisms remains the same.

Intoxicação natural por Clostridium botulinum tipo C: em grupo de aves domésticas

Clostridium botulinum causative of toxic infections due to toxin ingestion previously formed, occur in several ingestion species, mainly birds. In a poultry farm, located at São Paulo State, more than 3,000 birds have been attacked by this toxic infections, when the birds showed motionless, loss of weight, accelerated, breathing, resulting in death of the birds. The blood harvest for attainment of the serum and later necropsy was carried out throwgh the following samples: liver, gizzard, crop, feed, water and litter. After that, the toxin was extracted by gelatin-phosphate buffer and inoculation in mice, isolation of the agent in Blood. Ágar and Reinforced Clostridium Ágar and neutralization of toxin determine its type. The inoculation in mice showed positive results in samples of liver, gizzad, crop and symptoms like was waist, and death of the birds by limp paralysis. The colonies that have been isolated, suspected of Clostridium botulinum showed expected results and further analysis revealed positive results to botulinical toxin type C. The farms have to pay attention in the routine jobs, choicer, and elimination of carcass because they are essential to keep this problem away.

Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites

Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Rv1712 locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially Phosphorylates dCMP

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Streptococcus mutans attachment on a cast titanium surface

This study examined by means of scanning electron microscopy (SEM), the attachment of Streptococcus mutans and the corrosion of cast commercially pure titanium, used in dental dentures. The sample discs were cast in commercially pure titanium using the vacuum-pressure machine (Rematitan System). The surfaces of each metal were ground and polished with sandpaper (#300-4000) and alumina paste (0.3 μm). The roughness of the surface (Ra) was measured using the Surfcorder rugosimeter SE 1700. Four coupons were inserted separately into Falcon tubes contained Mueller Hinton broth inoculated with S. mutans ATCC 25175 (109 cuf) and incubated at 37 °C. The culture medium was changed every three days during a 365-day period, after which the falcons were prepared for observations by SEM. The mean Ra value of CP Ti was 0.1527 μm. After S. mutans biofilm removal, pits of corrosion were observed. Despite the low roughness, S. mutans attachment and biofilm formation was observed, which induced a surface corrosion of the cast pure titanium.

The effect of chloride ions and A. ferrooxidans on the oxidative dissolution of the chalcopyrite evaluated by electrochemical noise analysis (ENA)

It is believed that the dissolution of chalcopyrite (CuFeS2) in acid medium can be accelerated by the addition of Cl- ions, which modify the electrochemical reactions in the leaching system. Electrochemical noise analysis (ENA) was utilized to evaluate the effect of the Cl- ions and Acidithiobacillus ferrooxidans on the oxidative dissolution of a CPE-chalcopyrite (carbon paste electrode modified with chalcopyrite) in acid medium. The emphasis was on the analysis of the admittance plots (Ac) calculated by ENA. In general, a stable passive behavior was observed, mainly during the initial stages of CPE-chalcopyrite immersion, characterized by a low passive current and a low dispersion of the Ac plots, mainly after bacteria addition. This can be explained by the adhesion of bacterial cells on the CPE-chalcopyrite surface acting as a physical barrier. The greater dispersions in the Ac plots occurred immediately after the Cl- ions addition, in the absence of bacteria characterizing an active-state. In the presence of bacteria the addition of Clions only produced some effect after some time due to the barrier effect caused by bacteria adhesion. © (2009) Trans Tech Publications.

Leaching of Ni and Cu from mine wastes (tailings and slags) using acid solutions and A. ferrooxidans

The objective of this work is to evaluate the acidic and biological leaching of tailings containing Ni/Cu from a flotation and smelting plant. Acidithiobacillus ferrooxidans, strain LR, was used for bioleaching at pH 1.8 and chemical controls were run parallel to that. The acidic leaching was done within 48 hours at pH 0.5 and 1.0. In the slag inoculated flasks the redox potential was high (600 mV), thus indicating oxidative bacterial activity, however, the obtained results after 15 days showed only around 13% Ni and 8% Cu extractions, which were not different to those of the controls. For the flotation tailings bioleaching extractions were approximately 45% for Ni and 16% for Cu while differing figures were obtained for the chemical controls. These were 30% and 12% respectively. Here we could observe that the presence of bacterial activity led to a higher solubility of Ni. Acid leaching of slag showed higher nickel and copper extractions: 56% and 24% respectively at pH 0.5 and 21% and 11% at pH 1.0. However, the acid consumption was 320 and 150 Kg/ton of slag, respectively, both much higher than in bacterial assays. These results indicated that Ni and Cu solubilization from the slag is acid dependent no matter the redox potential or ferric iron concentration of the leaching solution. For flotation tailings, acid treatment showed extractions of 23% for Ni and 16% for copper at pH 0.5 and 22% and 28%, respectively at pH 1.0. The acid consumption was also higher: 220 and 120 Kg/ton, at pH 0.5 and 1.0, respectively. Based on own findings we could observe that acid leaching is found to be more effective for slag, though the acid consumption is much higher, while for the flotation tailings, bacterial leaching seems to be the best alternative. © (2009) Trans Tech Publications.

Antibacterial activity of alkyl gallates against Xanthomonas citri subsp. citri

The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a seriousdisease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread ofX. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). Thetreatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a commontarget involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection. © 2013, American Society for Microbiology.

Avaliação comparativa entre PCR gênero-específica, PCR espécie-específica e nested PCR espécie-específica no diagnóstico da infecção por Brucella ovis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diversidade bacteriana em piscicultura neotropical

Pós-graduação em Microbiologia Agropecuária - FCAV