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Our objective is to provide an optimistic strategy for reversing soil degradation by increasing public and private research efforts to understand the role of soil biology, particularly microbiology, on the health of our world's soils. We begin by defining soil quality/soil health (which we consider to be interchangeable terms), characterizing healthy soil resources, and relating the significance of soil health to agroecosystems and their functions. We examine how soil biology influences soil health and how biological properties and processes contribute to sustainability of agriculture and ecosystem services. We continue by examining what can be done to manipulate soil biology to: (i) increase nutrient availability for production of high yielding, high quality crops; (ii) protect crops from pests, pathogens, weeds; and (iii) manage other factors limiting production, provision of ecosystem services, and resilience to stresses like droughts. Next we look to the future by asking what needs to be known about soil biology that is not currently recognized or fully understood and how these needs could be addressed using emerging research tools. We conclude, based on our perceptions of how new knowledge regarding soil biology will help make agriculture more sustainable and productive, by recommending research emphases that should receive first priority through enhanced public and private research in order to reverse the trajectory toward global soil degradation.

To ensure survival, most bacteria must acquire iron, a resource that is sequestered by mammalian hosts. Pathogenic bacteria have therefore evolved intricate systems to sense iron limitation and regulate gene expression appropriately. We used a pan-Neisseria microarray to examine genes regulated in Neisseria gonorrhoeae in response to iron availability in defined medium. Overall, 203 genes varied in expression, 109 up-regulated and 94 down-regulated by iron deprivation. In iron-replete medium, genes essential to rapid bacterial growth were preferentially expressed, while iron transport functions, and predominantly genes of unknown function, were expressed in low-iron medium. Of those TonB-dependent proteins encoded in the FA1090 genome with unknown ligand specificity, expression of three was not controlled by iron availability, suggesting that these receptors may not be high-affinity transporters for iron-containing ligands. Approximately 30% of the operons regulated by iron appeared to be directly under control of Fur. Our data suggest a regulatory cascade where Fur indirectly controls gene expression by affecting the transcription of three secondary regulators. Our data also suggest that a second MerR-like regulator may be directly responding to iron availability and controlling transcription independent of the Fur protein. Comparison of our data with those recently published for Neisseria meningitidis revealed that only a small portion of genes were found to be similarly regulated in these closely related pathogens, while a large number of genes derepressed during iron starvation were unique to each organism.

Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.Listeria monocytogenes is the causative agent of listeriosis, a food-borne disease with clinical presentations that include febrile gastroenteritis, encephalitis, meningitis, septicemia, and spontaneous abortion (7). Listeriosis infections are associated with high hospitalization (92%) and mortality (20 to 30%) rates and account for over one-quarter of all deaths attributable to known food-borne pathogens (12, 24). L. monocytogenes is widely distributed in the environment, forms biofilms, grows at refrigeration temperatures, and is relatively resistant to acid and high salt concentrations (23, 48). These characteristics enable L. monocytogenes to persist for extended periods in food-processing environments and make L. monocytogenes contamination of ready-to-eat (RTE) foods a significant concern. Accordingly, regulatory agencies have applied a zero tolerance policy for L. monocytogenes contamination in RTE foods, and L. monocytogenes has been a leading cause of food recalls due to microbial adulteration.Molecular subtyping is a critical component of L. monocytogenes outbreak detection and epidemiological investigations, which are complicated by the long incubation time for invasive listeriosis and the difficulty in identifying appropriate controls for case-control studies (44). Pulsed-fiel...

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies-temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)-to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.The acquisition of iron is essential for bacterial pathogenesis. Because iron is insoluble in aqueous environments at neutral pH and has the potential to produce damaging free radicals, the mammalian host tightly sequesters iron (1, 51), lowering the level of free iron available to invading microorganisms to well below what is needed to survive (34). Since survival in the host and hence virulence is dependent on the acquisition of iron, pathogenic bacteria must be able to sense iron availability and globally regulate the transcription of iron acquisition genes. For many gram-positive and gram-negative organisms, including the sexually transmitted disease pathogen Neisseria gonorrhoeae, the crucial balance between acquiring enough iron to grow and avoiding the toxic effects of excess iron is controlled by the ferric uptake regulator (Fur) protein (12, 27, 49). Typically, Fur acts as a transcriptional repressor by binding as a dimer, along with ferrous iron as a corepressor, to regulatory Fur box (FB) sequences in the promoters of iron-regulated genes under iron-rich growth conditions. As intracellular iron stores are depleted, the Fur-Fe 2ϩ dimer dissociates from the promoter, allowing the entry of RNA polymerase and subsequent transcription (22). Fur acts as a global regulator controlling not only the expression of iron acquisition...

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.

a b s t r a c tIt has been demonstrated that soil amended with biochar, designed specifically for use as a soil conditioner, results in changes to the microbial populations that reside therein. These changes have been reflected in studies measuring variations in microbial activity, biomass, and community structure. Despite these studies, very few experiments have been performed examining microbial genes involved in nutrient cycling processes. Given the paucity of research in this area, we designed a 6 month study in a Portneuf subsoil treated with three levels (1%, 2%, and 10% w/w ratio) of a biochar pyrolyzed from switchgrass (Panicum virgatum) at 350 • C and steam activated at 800 • C to measure the abundances of five genes involved in N cycling. Gene abundances were measured using qPCR, with relative abundances of these genes calculated based on measurement of the 16S rRNA gene. At the end of the 6 month study, all measured genes showed significantly greater abundances in biochar amended treatments as compared to the control. In soil amended with 10% biochar, genes involved in nitrogen fixation (nifH), and denitrification (nirS), showed significantly increased relative abundances. Lastly, gene abundances and relative abundances correlated with soil characteristics, in particular NO 3 -N, % N and % C. These results confirm that activated switchgrass-derived biochar, designed for use as a soil conditioner, has an impact on the treated soils microbial communities. We therefore suggest that future use of biochar as a soil management practice should take into account not only changes to the soil's physiochemical properties, but its biological properties as well.Published by Elsevier B.V.

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