Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH).

BACKGROUND: In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. RESULTS: We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. CONCLUSION: We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

Atmospheric emission of reactive nitrogen during biofuel ethanol production.

This paper evaluates emissions to the atmosphere of biologically available nitrogen compounds in a region characterized by intensive sugar cane biofuel ethanol production. Large emissions of NH3 and NOx, as well as particulate nitrate and ammonium, occur at the harvest when the crop is burned, with the amount of nitrogen released equivalent to approximately 35% of annual fertilizer-N application. Nitrogen oxides concentrations show a positive association with fire frequency, indicating that biomass burning is a major emission source, with mean concentrations of NOx doubling in the dry season relative to the wetseason. During the dry season biomass burning is a source of NH3, with other sources (wastes, soil, biogenic) predominant during the wet season. Estimated NO2-N, NH3-N, NO3- -N and NH4+ -N emission fluxes from sugar cane burning in a planted area of ca. 2.2 x 10(6) ha are 11.0, 1.1, 0.2, and 1.2 Gg N yr(-1), respectively.

Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections.

Proteus mirabilis is an important uropathogen that can cause complicated urinary tract infections (UTI). It produces several types of fimbriae, including mannose-resistant Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF). Previously, we determined that these fimbriae affect the ability of P. mirabilis to colonize the urinary tract. The objective of this study was to analyse the effect of the simultaneous lack of P. mirabilis MR/P and PMF fimbriae in UTI pathogenesis. A double mutant lacking both fimbriae was generated by allelic replacement mutagenesis. This mutant was characterized genetically and phenotypically, and tested using an in vitro uroepithelial cell adhesion assay and the ascending UTI murine model. In vitro adhesion to uroepithelial cells by the P. mirabilis pmfA/mrpA-D mutant was reduced when compared with the wild-type, although no significant differences were observed when it was compared with the single mrpA-D and pmfA mutants. However, in vivo assays showed that colonization of kidneys and bladders by the P. mirabilis pmfA/mrpA-D mutant was significantly reduced when compared with the wild-type and both single mutants. These results indicate that, although redundancy can occur, MR/P and PMF fimbriae have specific and additive roles in P. mirabilis UTI.

Trace metal concentrations and water solubility in size-fractionated atmospheric particles and influence of road traffic.

The abundance and the behavior of metals (Al, Ti, Mn, Fe, Co, Ni, Cu, Zn, Se, Ag, Cd, Sn, Ba, Pt, Hg, and Pb) and ions (Na+ K+ Mg2+ Ca2+, NH4+, Cl-, NO3-, SO4(2-), PO4(3-), and oxalate) in size-fractionated atmospheric particulate matter (PM) were studied in the U. K. and Ireland at four observation sites simulating extreme degrees of vehicular-traffic influence in the environment. Trace metals in urban PM showed distinct types of size-fractionated behavior depending on the particle sources from which they originate. In coarse PM (1.5 < Dp < 3.0 microm) the concentrations of copper, barium, and iron correlated closely across over 2 orders of magnitude in urban air, which is seen as evidence that major portions of transition metals (Cu, Ba, Fe, and Mn) are released through abrasive vehicular emissions, particularly the wear of brake linings. Further results are strongly indicative of a decoupling of coarse iron and calcium, the former arising predominantly from vehicles, the latter from soil resuspension. In fine PM (Dp < 0.5 microm), several combustion and secondary sources of particulates were identified, but these were much less unique in terms of elemental fingerprints. An analysis of the water solubility of trace metals yielded that solubility varies considerably with element and, to a lesser extent, with particle size. Notable differences were found to the elemental water solubilities determined in previous work, partially explained by differences in extraction procedures.

Influence of agricultural biomass burning on aerosol size distribution and dry deposition in southeastern Brazil.

The size distributed composition of ambient aerosols is used to explore seasonal differences in particle chemistry and to show that dry deposition fluxes of soluble species, including important plant nutrients, increase during periods of biomass (sugar cane trash) burning in São Paulo State, Brazil. Measurements were made at a single site centrally located in the State's sugar cane growing region but away from the immediate vicinity of burns, so that the airsampled was representative of the regional background. Calculation of ion equivalent balances showed that during burning periods smaller particles (Aitken and accumulation modes) were more acidic, containing higher concentrations of SO4(2-), oxalate, NO3-, HCOO-, CH3COO-, and CI-, but insufficient NH4+ and K+ to achieve neutrality. Larger particles showed an anion deficit due to the presence of unmeasured ions and comprised resuspended dusts modified by accumulation of nitrate, chloride, and organic anions. Increases of resuspended particles during the burning season were attributed to release of earlier deposits from the surfaces of burning vegetation as well as increased vehicle movement on unsurfaced roads. During winter months the relative contribution of combined emissions from road transport and industry diminished due to increased emissions from biomass combustion and other activities specifically associated with the harvest period. Positive increments in annual particulate dry deposition fluxes due to higher fluxes during the sugar cane harvest were 44.3% (NH4+), 42.1% (K+), 31.8% (Mg2+), 30.4% (HCOO-), 12.8% (CI-), 6.6% (CH3COO-), 5.2% (Ca2+), 3.8% (SO4(2-)), and 2.3% (NO3-). Na+ and oxalate fluxes were seasonally invariant. Annual aerosol dry deposition fluxes (kg ha(-1)) were 0.5 (Na+), 0.25 (NH4+), 0.39 (K+), 0.51 (Mg2+), 3.19 (Ca2+), 1.34 (Cl-), 4.47 (NO3-), 3.59 (SO4(2-)), 0.58 (oxalate), 0.71 (HCOO-), and 1.38 (CH3COO-). Contributions of this mechanism to combined aerosol dry deposition and precipitation scavenging (inorganic species, excluding gaseous dry deposition) were 31% (Na+), 8% (NH4+), 26% (K+), 63% (Mg2+), 66% (Ca2+), 32% (Cl-), 33% (NO3-), and 36% (SO4(2-)).

Bordetella pertussis risA, but not risS, is required for maximal expression of Bvg-repressed genes.

Expression of virulence determinants by Bordetella pertussis, the primary etiological agent of whooping cough, is regulated by the BvgAS two-component regulatory system. The role of a second two-component regulatory system, encoded by risAS, in this process is not defined. Here, we show that mutation of B. pertussis risA does not affect Bvg-activated genes or proteins. However, mutation of risA resulted in greatly diminished expression of Bvg-repressed antigens and decreased transcription of Bvg-repressed genes. In contrast, mutation of risS had no effect on the expression of Bvg-regulated molecules. Mutation of risA also resulted in decreased bacterial invasion in a HeLa cell model. However, decreased invasion could not be attributed to the decreased expression of Bvg-repressed products, suggesting that mutation of risA may affect the expression of a variety of genes. Unlike the risAS operons in B. parapertussis and B. bronchiseptica, B. pertussis risS is a pseudogene that encodes a truncated RisS sensor. Deletion of the intact part of the B. pertussis risS gene does not affect the expression of risA-dependent, Bvg-repressed genes. These observations suggest that RisA activation occurs through cross-regulation by a heterologous system.

NO2 emissions from agricultural burning in São Paulo, Brazil.

We report here on the application of a compact ultraviolet spectrometer to measurement of NO2 emissions from sugar cane field burns in São Paulo, Brazil. The time-resolved NO2 emission from a 10 ha plot peaked at about 240 g (NO2) s(-1), and amounted to a total yield of approximately 50 kg of N, or about 0.5 g (N) m(-2). Emission of N as NOx (i.e., NO + NO2) was estimated at 2.5 g (N) m(-2), equivalent to 30% of applied fertilizer nitrogen. The corresponding annual emission of NOx nitrogen from São Paulo State sugar cane burning was >45 Gg N. In contrast to mechanized harvesting, which does not require prior burning of the crop, manual harvesting with burning acts to recycle nitrogen into surface soils and ecosystems.

Evaluation of a novel passive sampling technique for monitoring volcanogenic hydrogen sulfide.

A novel, low-cost passive sampling procedure for monitoring of volcanogenic hydrogen sulfide is reported. The technique is based on absorption of H2S onto treated sections of photographic paper, which are housed in plastic film canisters during exposure. The H2S reacts with silver halide in the photographic paper, causing a colour-change reaction from white, through brown, to black, depending on concentration of atmospheric H2S. The sampler is sensitive to << 30 ppb to approximately 1000 ppb of H2S. Here we present results from a series of optimization and quantification experiments. An active sampling procedure for rapid H2S measurement is also reported, based on absorption of H2S onto Whatman No. 41 cellulose filters treated with silver nitrate, and was shown to be quantitative using a single filter at flow rates <1.0 L min(-1) for collection of <200 microg of sulfide (as H2S). Determination of sulfide collected on the substrates was performed using a rapid flow-injection technique based on the fluorescence quenching of fluorescein mercuric acetate (FMA) by sulfide. This was optimized at a FMA concentration of 8 mg L(-1), at which 100% quenching was obtained at a solution sulfide concentration of 3 mg L(-1).

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited. Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens. The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S. suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds. Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell. Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene. Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates. In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide. There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S. suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia.

Identification and characterisation of hyaluronate lyase from Streptococcus suis.

Hyaluronate lyase, which catalyses the degradation of hyaluronic acid (HA), has been described from several pathogenic streptococcal species. We describe, for the first time, identification and purification of hyaluronate lyase from the zoonotic pig pathogen Streptococcus suis. We have cloned the hyaluronate lyase gene from S. suis and used it to generate an allelic replacement knock-out mutant of S. suis serotype 7 that can no longer biosynthesise the enzyme. Interestingly, a limited strain survey indicates that hyaluronate lyase activity is not present in all disease isolates of S. suis. Polyclonal anti-hyaluronate lyase anti-serum raised against our recombinant hyaluronate lyase has been used in Western blots, showing that hyaluronate lyase activity is always associated with the presence of protein of the expected size, whereas lack of hyaluronate lyase activity is due to truncation or absence of the enzyme. We show that hyaluronate lyase activity is required for S. suis to use HA polymer as a carbon source and that supplying exogenous recombinant hyaluronate lyase to all S. suis strains tested allowed fermentation of the resultant HA breakdown products.

Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice.

Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Self-organization and the mind-brain problem

The problem of the relationship between mind and brain is discussed as a particular case of the problem of the relationship between mind and body, itself a product which results from adoption of the Cartesian paradigm. Following the procedures of Ryle(1) we will use, as a working hypothesis, the supposition that this problem might be dissolved through careful examination of its presuppositions. A possible way of approaching the relationship between mind and body using the theory of self-organization will be outlined. Our discussion will proceed as follows: 1) History of the problem of the mind/body relationship in the Cartesian paradigm; 2) proposed solutions to the mind/brain problem and some of their limitations; 3) Ryle's proposal for a dissolution of the mind/body problem; 4) an outiline of the mind/body relationship from the perspective of self-organization theory.

A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases.

Copies of multi-gene family, named PRT1 (protease 1), encoding a subtilisin-like serine protease were cloned from the opportunistic fungal pathogen Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic clone and a cDNA clone of PRT1 from P. carinii f. sp. carinii revealed the presence of seven short introns. Several different domains were predicted from the deduced amino acid sequence: an N-terminal hydrophobic signal sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain (essential for proteolytic activity), a proline-rich domain, a serine/threonine-rich domain and a C-terminal hydrophobic domain. The catalytic domain showed high homology to other eukaryotic subtilisin-like serine proteases and possessed the three essential residues of the catalytic active site. Karyotypic analysis showed that PRT1 was a multi-gene family, copies of which were present on all but one of the P. carinii f. sp. carinii chromosomes. The different copies of the PRT1 genes showed nucleotide sequence heterogeneity, the highest level of divergence being in the proline-rich domain, which varied in both length and composition. Some copies of PRT1 were contiguous with genes encoding the P. carinii major surface glycoprotein.