Summary
Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two‐component system regulators CbrAB, NtrBC and ...PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C‐P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2‐aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant‐growth‐promoting rhizobacterium Pseudomonas putida BIRD‐1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR‐type regulator, termed AepR, upstream of the 2AEP transaminase‐phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate‐specific regulators in phosphonate metabolism.
Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series ...of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.
Footrot is an infectious bacterial disease of sheep that causes lameness. The causal agent is Dichelobacter nodosus. There is debate regarding the role of Fusobacterium necrophorum in disease ...initiation. This research used an observational longitudinal study of footrot, together with quantitative PCR (qPCR) of bacterial load of D. nodosus and F. necrophorum, to elucidate the roles of each species in the development of disease. All feet of 18 a priori selected sheep were monitored for five weeks assessing disease severity (healthy, interdigital dermatitis (ID) and severe footrot (SFR)) and bacterial load. A multinomial model was used to analyse these data.
Key unadjusted results were that D. nodosus was detected more frequently on feet with ID, whereas F. necrophorum was detected more frequently on feet with SFR. In the multinomial model, ID was associated with increasing log10 load of D. nodosus the week of observation (OR=1.28 (95% CI=1.08–1.53)) and the week prior to development of ID (OR=1.20 (95% CI=1.01–1.42). There was no association between log10 load2 of F. necrophorum and presence of ID (OR=0.99 (95% CI=0.96–1.02))). SFR was associated with increasing log10 load of D. nodosus the week before disease onset (OR=1.42 (95% CI=1.02–1.96)) but not once SFR had occurred. SFR was positively associated with log10 load2 of F. necrophorum once disease was present (OR=1.06 (95% CI=1.01–1.11)). In summary, there was an increased risk of increasing D. nodosus load the week prior to development of ID and SFR and during an episode of ID. In contrast, F. necrophorum load was not associated with ID before or during an episode, and was only associated with SFR once present. These results contribute to our understanding of the epidemiology of footrot and highlight that D. nodosus load plays the primary role in disease initiation and progression, with F. necrophorum load playing a secondary role. Further studies in more flocks and climates would be useful to confirm these findings. This study identifies that D. nodosus load is highest during ID. This supports previous epidemiological findings, which demonstrate that controlling ID is the most effective management strategy to prevent new cases of ID and SFR.
Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed ...species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.
In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of
complex (MTBC) is a major challenge affecting TB management. This ...study utilized comparative genomic analyses of MTBC lineages;
,
Lineages 5/6 and
to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by
, while
L5 & L6 reported 9.0% and 14.4%, respectively.
infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.
Advances in DNA sequencing technologies have drastically changed our perception of the structure and complexity of the plant microbiome. By comparison, our ability to accurately identify the ...metabolically active fraction of soil microbiota and its specific functional role in augmenting plant health is relatively limited. Important ecological interactions being performed by microbes can be investigated by analyzing the extracellular protein fraction. Here, we combined a unique protein extraction method and an iterative bioinformatics pipeline to capture and identify extracellular proteins (metaexoproteomics) synthesized in the rhizosphere of
spp. We first validated our method in the laboratory by successfully identifying proteins related to a host plant (
) and its bacterial inoculant, Pseudomonas putida BIRD-1. This identified numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients in P. putida. Next, we analyzed natural field-soil microbial communities associated with
L. (oilseed rape). By combining metagenomics with metaexoproteomics, 1,885 plant, insect, and microbial proteins were identified across bulk and rhizosphere samples. Metaexoproteomics identified a significant shift in the metabolically active fraction of the soil microbiota responding to the presence of
roots that was not apparent in the composition of the total microbial community (metagenome). This included stimulation of rhizosphere-specialized bacteria, such as
,
, and
, and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralization. Our metaproteomic assessment of the "active" plant microbiome at the field-scale demonstrates the importance of moving beyond metagenomics to determine ecologically important plant-microbe interactions underpinning plant health.
Plant-microbe interactions are critical to ecosystem function and crop production. While significant advances have been made toward understanding the structure of the plant microbiome, learning about its full functional role is still in its infancy. This is primarily due to an incomplete ability to determine
plant-microbe interactions actively operating under field conditions. Proteins are the functional entities of the cell. Therefore, their identification and relative quantification within a microbial community provide the best proxy for which microbes are the most metabolically active and which are driving important plant-microbe interactions. Here, we provide the first metaexoproteomics assessment of the plant microbiome using field-grown oilseed rape as the model crop species, identifying key taxa responsible for specific ecological interactions. Gaining a mechanistic understanding of the plant microbiome is central to developing engineered plant microbiomes to improve sustainable agricultural approaches and reduce our reliance on nonrenewable resources.
In soil, bioavailable inorganic orthophosphate is found at low concentrations and thus limits biological growth. To overcome this phosphorus scarcity, plants and bacteria secrete numerous enzymes, ...namely acid and alkaline phosphatases, which cleave orthophosphate from various organic phosphorus substrates. Using profile hidden Markov modeling approaches, we investigated the abundance of various non specific phosphatases, both acid and alkaline, in metagenomes retrieved from soils with contrasting pH regimes. This analysis uncovered a marked reduction in the abundance and diversity of various alkaline phosphatases in low‐pH soils that was not counterbalanced by an increase in acid phosphatases. Furthermore, it was also discovered that only half of the bacterial strains from different phyla deposited in the Integrated Microbial Genomes database harbor alkaline phosphatases. Taken together, our data suggests that these ‘phosphatase lacking’ isolates likely increase in low‐pH soils and future research should ascertain how these bacteria overcome phosphorus scarcity.
Phosphorus (P) is an essential element for all living organisms. Soil‐dwelling microorganisms are beneficial to plants as they can remineralize unavailable organic P, thus acting as biofertilizers. Our analyses revealed a significant reduction in the abundance of promiscuous organic P cleaving phosphatases in low‐pH soils, suggesting a potential reduction in their ability to remineralize P.
We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a ...flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.
Dichelobacter nodosus (D. nodosus) is the causative agent of footrot in sheep; one of the most important health and welfare issues of sheep worldwide. For control programmes to be effective, it is ...essential that the transmission cycle of D. nodosus is understood and bacterial reservoirs in the environment are better defined. This study evaluated the survival of D. nodosus in different soils using soil microcosms. Cultivation independent and dependent methods were used to detect D. nodosus over 40 days from seeding in soil. A D. nodosus specific probe was used for quantification by qPCR and viability was assessed by cell permeability to an intercalating dye, PMA, and by culture. Survival varied dramatically depending on soil type, matric potential (MP) and temperature. Our findings indicate that D. nodosus survival was higher at 5 °C compared with 25 °C in all soils and significantly longer at both temperatures in clay soil (>44% clay) compared with other soil types. Survival under all conditions was longer than 30 days for both culture independent and dependent methods, this is substantially longer than previous studies and, if this is an infectious dose, longer than the current recommendation of resting a field for 14 days to prevent onward infection.
•Viable Dichelobacter nodosus was detected for 40 days off host in ventilated soil microcosms.•D. nodosus survival rate was affected by matric potential, temperature and soil type.•D. nodosus survived for 40 days both at 5 °C and 25 °C in four contrasting soils.•D. nodosus survival was higher at 5 °C compared with 25 °C in all soils.•D. nodosus survival was significantly longer in clay soil (>44% clay).
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