Aluminum toxicity in acid soils severely limits crop productivity through inhibition of root growth and, consequently, shoot development. Several Arabidopsis mutants were previously identified as having roots with Al hypersensitivity, suggesting that these represent deleterious mutations affecting genes required for either Al tolerance or resistance mechanisms. For this report, the als1-1 mutant was chosen for further characterization. The phenotype of als1-1 is most obviously presented in Al challenged roots, as evidenced by exaggerated root growth inhibition in conjunction with increased expression of Al-responsive genes compared to wt. Using a map-based cloning approach, the als1-1 mutation was isolated and found to represent a deleterious amino acid substitution in a previously uncharacterized half type ABC transporter, At5g39040, which is expressed in a non-Al dependent manner in all organs tested. GUS-dependent analyses revealed that ALS1 expression is primarily localized to the root tip and the vasculature throughout the plant. Concomitant with this, an ALS1: GFP fusion accumulates at the vacuolar membrane of root cells, indicating that ALS1 may be important for intracellular movement of some substrate, possibly chelated Al, as part of a mechanism of Al sequestration.

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion.

Aluminum (Al) toxicity is a global problem severely limiting agricultural productivity in acid-soil regions comprising upwards of 50% of the world's arable land [1, 2]. Although Al-exclusion mechanisms have been intensively studied [3-9], little is known about tolerance to internalized Al, which is predicted to be mechanistically complex because of the plethora of predicted cellular targets for Al(3+)[2, 10]. An Arabidopsis mutant with Al hypersensitivity, als3-1, was found to represent a lesion in a phloem and root-tip-localized factor similar to the bacterial ABC transporter ybbm, with ALS3 likely responsible for Al transfer from roots to less-sensitive tissues [10-12]. To identify mutations that enhance mechanisms of Al resistance or tolerance, a suppressor screen for mutants that mask the Al hypersensitivity of als3-1 was performed [13]. Two allelic suppressors conferring increased Al tolerance were found to represent dominant-negative mutations in a factor required for monitoring DNA integrity, AtATR[14-17]. From this work, Al-dependent root-growth inhibition primarily arises from DNA damage coupled with AtATR-controlled blockage of cell-cycle progression and terminal differentiation because of loss of the root-quiescent center, with mutations that prevent response to this damage resulting in quiescent-center maintenance and sustained vigorous growth in an Al-toxic environment.

BackgroundLyme disease, caused by various species of Borrelia, is transmitted by Ixodes ticks in North America and Europe. Studies have shown the genotype of Borrelia burgdorferi sensu stricto (s.s.) or the species of B. burgdorferi sensu lato (s.l.) affects the ability of the bacteria to cause local or disseminated infection in humans.Methodology/Principal FindingsWe used a multilocus PCR electrospray mass spectrometry assay to determine the species and genotype Borrelia from ticks collected in New York, Connecticut, Indiana, Southern Germany, and California and characterized isolates from parts of the United States and Europe. These analyses identified 53 distinct genotypes of B. burgdorferi sensu stricto with higher resolution than ospC typing. Genotypes of other members of the B. burgdorferi sensu lato complex were also identified and genotyped including B. afzelii, B. garinii, B. lusitaniae, B. spielmanii, and B. valaisiana. While each site in North America had genotypes unique to that location, we found genotypes shared between individual regions and two genotypes found across the United States. Significant B. burgdorferi s.s. genotypic diversity was observed between North America and Europe: only 6.6% of US genotypes (3 of 45) were found in Europe and 27% of the European genotypes (3 of 11) were observed in the US. Interestingly, 39% of adult Ixodes scapularis ticks from North America were infected with more than one genotype of B. burgdorferi s.s. and 22.2% of Ixodes ricinus ticks from Germany were infected with more than one genotype of B. burgdorferi s.l.Conclusions/SignificanceThe presence of multiple Borrelia genotypes in ticks increases the probability that a person will be infected with more than one genotype of B. burgdorferi, potentially increasing the risks of disseminated Lyme disease. Our study indicates that the genotypic diversity of Borrelia in ticks in both North America and Europe is higher then previously reported and can have potential clinical consequences.

Infection rates of Ixodes ticks with Borrelia miyamotoi in Europe and the United States vary greatly based upon location.

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States.

28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial 28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the 28 -dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a ؊35 element recognized by chlamydial 28 RNA polymerase that resembles the consensus ؊35 sequence. Within the ؊10 element, however, chlamydial 28 RNA polymerase showed a striking preference for a CGA sequence at positions ؊12 to ؊10 rather than the longer consensus ؊10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli 28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the ؊10 promoter element recognized by 28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that 28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11-or 12-nt spacer equally well. Altogether, we found very similar results for 28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred 28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n 11/12 -ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n 11/12 is a spacer of 11 or 12 nt.