Microbial interactions in soil

Our view on the diversity and distribution of soil microbiota has expanded and continues to do so, driven by high-throughput sequencing technologies, but comparatively little is known about how these organisms affect each other. Bacteria, archaea, fungi, protists and their respective viruses impact each other through a range of beneficial and deleterious interactions, and thereby the soil ecosystem [1]. Modern microbiology, such as agriculture, has been shaped by the mono-culture paradigm, and the secrets of cellular function have been uncovered using a single culture approach. For decades, microbiologists have been trained to obtain and study “pure cultures”, clonal lineages able to grow rapidly on protein-rich laboratory media. In contrast, most microorganisms occur in soil and aquatic environments, surrounded by a myriad of life forms from bacteria, fungi and protists to insects, occurring at high densities amid sparse nutrient availability [2,3]. Bacteria contribute 70 Gt of the 550 Gt of global carbon biomass, together with 7 Gt from Archaea, 12 from fungi and 4 from protists. The terrestrial microbial biomass is estimated to be composed of 7 Gt carbon of bacteria, 0.5 of archaea, 12 of fungi and 1.6 of protists [4], so bacteria constitute the largest part of microbiota, not only by number, but also by biomass. In contrast, humans, the hosts to the most studied microbial ecosystem, make up only 0.06 Gt of carbon.https://www.mdpi.com/journal/microorganismsam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with (H2O)-O-18

Current limitations in culture-based methods have lead to a reliance on culture- independent approaches, based principally on the comparative analysis of primary semantides such as ribosomal gene sequences. DNA can be remarkably stable in some environments, so its presence does not indicate live bacteria, but extracted ribosomal RNA (rRNA) has previously been viewed as an indicator of active cells. Stable isotope probing (SIP) involves the incorporation of heavy isotopes into newly synthesized nucleic acids, and can be used to separate newly synthesized from existing DNA or rRNA. H2 18O is currently the only potential universal bacterial substrate suitable for SIP of entire bacterial communities. The aim of our work was to compare soil bacterial community composition as revealed by total versus SIP-labeled DNA and rRNA. Soil was supplemented with H2 18O and after 38 days the DNA and RNA were co-extracted. Heavy nucleic acids were separated out by CsCl and CsTFA density centrifugation. The 16S rRNA gene pools were characterized by DGGE and pyrosequencing, and the sequence results analyzed using mothur. The majority of DNA (~60%) and RNA (~75%) from the microcosms incubated with H2 18O were labeled by the isotope. The analysis indicated that total and active members of the same type of nucleic acid represented similar community structures, which suggested that most dominant OTUs in the total nucleic acid extracts contained active members. It also supported that H2 18O was an effective universal label for SIP for both DNA and RNA. DNA and RNA-derived diversity was dissimilar. RNA from this soil more comprehensively recovered bacterial richness than DNA because the most abundant OTUs were less numerous in RNA than DNAderived community data, and dominant OTU pools didn’t mask rare OTUs as much in RNA.SD00H296-081HG from the South Dakota Agricultural Experiment Station to V. S. B. E. A. R. was supported by a fellowship from the NASA South Dakota Space Grant Consortium. We acknowledge use of the SDSU-Functional Genomics Core Facility, supported by NSF/EPSCoR Grant No. 0091948, the South Dakota 2010 Drought Initiative, and the South Dakota Agricultural Experiment Station.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2045-8827hb201

Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.This research was supported by a grant from the Water Research Commission of South Africa (WRC K5/1276) to VSB and SNV, and National Research Foundation (NRF) grant 2046811 to VSB. LMS was supported by a scholarship from the NRF of South Africa.http://www.iwaponline.com/jwh/default.htmhb201

Diversity of free-living nitrogen fixing Streptomyces in soils of the badlands of South Dakota

Biological Nitrogen Fixation is critical for ecosystem productivity. Select members of Bacteria and Archaea express a nitrogenase enzyme complex that reduces atmospheric nitrogen to ammonia. Several nitrogen fixing bacteria form symbiotic associations with plants, but freeliving diazotrophs also contribute a substantial amount of nitrogen to ecosystems. The aim of this study was to isolate and characterize free-living diazotrophs in arid lands of South Dakota Badlands. Samples were obtained from sod tables and the surrounding base in spring and fall. Diazotrophs were isolated on solid nitrogen free medium (NFM) under hypoxic conditions, and their16S rRNA and nifH genes sequenced. nifH was also amplified directly from soil DNA extracts. The 16S rRNA gene data indicated a diversity of putative free-living diazotrophs across 4 phyla (Actinomycetes, Proteobacteria, Bacteroidetes, and Firmicutes), but ~50% of these clustered with Streptomyces. These Streptomyces isolates grew in liquid NFM in an ammoniadepleted environment. Only 5 of these yielded a nifH gene product using the PolF/PolR primer set. Four of these aligned with nifH of the cyanobacteria Scytonema and Nostoc, and the other one aligned with nifH of Bradyrhizobium. Six selected Streptomyces isolates, three of which were nifH positive by PCR, all indicated 15N2 incorporation, providing strong support of nitrogen fixation. All nifH amplicons from soil DNA extract resembled Cyanobacteria. This is the first known report of diazotrophic Streptomyces, other than the thermophilic, autotrophic S. thermoautotrophicus. nifH genes of these Streptomyces were related to those from Cyanobacteria. It is possible that the cyanobacteria-like nifH amplicons obtained from soil DNA were associated with Streptomyces.BD was supported by a fellowship from the South Dakota Agricultural Experiment Station. This work was supported by the South Dakota Agricultural Experiment Station. We acknowledge use of the SDSU-Functional Genomics Core Facility, supported by NSF/EPSCoR Grant No. 0091948, the South Dakota 2010 Drought Initiative, and the South Dakota Agricultural Experiment Station.http://www.elsevier.com/locate/micres2018-01-31hb2017Microbiology and Plant Patholog

Spatio temporal influence of isoflavonoids on bacterial diversity in the soybean rhizosphere

High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. However, it is unclear if and how specific rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids, a major component of soybean rhizodeposits, using RNA interference in hairy-root composite plants, and examined changes in rhizosphere bacteriome diversity. We used successive sonication to isolate soil fractions from different rhizosphere zones at two different time points and analyzed denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene amplicons. Extensive diversity analysis of the resulting spatio temporal profiles of soybean bacterial communities indicated that, indeed, isoflavonoids significantly influenced soybean rhizosphere bacterial diversity. Our results also suggested a temporal gradient effect of rhizodeposit isoflavonoids on the rhizosphere. However, the hairy-root transformation process itself significantly altered rhizosphere bacterial diversity, necessitating appropriate additional controls. Gene silencing in hairy-root composite plants combined with successive sonication is a useful tool to determine the spatio temporal effect of specific rhizodeposit compounds on rhizosphere microbial communities.http://apsjournals.apsnet.org/loi/mpmi2016-01-31hb201

The microbial nitrogen cycling, bacterial community composition, and functional potential in a natural grassland are stable from breaking dormancy to being dormant again

DATA AVAILABILITY STATEMENT : The raw data is available on NCBI’s Sequence Read Archive (SRA) database under BioProject: PRJNA803487.SUPPLEMENTARY MATERIALS : FIGURE S1: Soil textures of the sampling sites; FIGURE S2: Distribution of plant groups across the six study sites; FIGURE S3: Plant species distribution across the study sites. The legume species are represented by asterisk (*) on the legend. The abbreviations represent: BRIN, Bromus inermis; POPR, Poa pratensis; PHAR, Phalaris arundinacea; CA, Carex sp.; NA, Nassella sp.; ANGE, Andropogon gerardii; SCSC, Schizachyrium scoparium; BOCU, Bouteloua curtipendula; DIOL, Dichanthelium oligosanthes; CIFL, Cirsium flodmanii; ASSP, Asclepias speciose; SOMI, Solidago missouriensis; HEMA, Helianthus maximiliani; ANCA, Anemone canadensis; SOCA, Solidago canadensis; GLLE, Glycyrrhiza lepidota; PH, Physalis sp.; AMCA*, Amorpha canescens; DAPU*, Dalea purpurea; PE*, Pediomelum sp.; RO, Rosa sp.; FIGURE S4: Soil chemical properties across sampling sites within the sampling time points with Kruskal–Wallis test results; FIGURE S5: Alpha-diversity of the bacterial communities across 6 months. (a) Shannon diversity and (b) Pielou Evenness.; FIGURE S6: Taxa differences across at least one time point across the seasons (p < 0.01). The names of the phyla shown here are based on the taxonomic profile downloaded from the Greengenes database, however, some of the phylum names have recently been changed [38]; TABLE S1. G-Block solutions; TABLE S2. Primers used for nitrogen cycle genes. SUPPLEMENTARY DATA S3. R 2 values of the qPCR assays. SUPPLEMENTARY DATA S4. OTU table.The quantity of grass-root exudates varies by season, suggesting temporal shifts in soil microbial community composition and activity across a growing season. We hypothesized that bacterial community and nitrogen cycle-associated prokaryotic gene expressions shift across three phases of the growing season. To test this hypothesis, we quantified gene and transcript copy number of nitrogen fixation (nifH), ammonia oxidation (amoA, hao, nxrB), denitrification (narG, napA, nirK, nirS, norB, nosZ), dissimilatory nitrate reduction to ammonia (nrfA), and anaerobic ammonium oxidation (hzs, hdh) using the pre-optimized Nitrogen Cycle Evaluation (NiCE) chip. Bacterial community composition was characterized using V3-V4 of the 16S rRNA gene, and PICRUSt2 was used to draw out functional inferences. Surprisingly, the nitrogen cycle genes and transcript quantities were largely stable and unresponsive to seasonal changes. We found that genes and transcripts related to ammonia oxidation and denitrification were different for only one or two time points across the seasons (p < 0.05). However, overall, the nitrogen cycling genes did not show drastic variations. Similarly, the bacterial community also did not vary across the seasons. In contrast, the predicted functional potential was slightly low for May and remained constant for other months. Moreover, soil chemical properties showed a seasonal pattern only for nitrate and ammonium concentrations, while ammonia oxidation and denitrification transcripts were strongly correlated with each other. Hence, the results refuted our assumptions, showing stability in N cycling and bacterial community across growing seasons in a natural grassland.The South Dakota Agricultural Experiment Station.https://www.mdpi.com/journal/microorganismsam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Fingerprinting of commercially available water treatment bactericides in South Africa

Eighteen dominant isolates from water-cooling systems were exposed to 50mg/l of commercially available bactericides, and the kill percentage was determined after 6 h. Application costs of all bactericides giving an average kill percentage of over 90%, were compared. Low cost bactericides were re-evaluated at cost-equivalent concentrations. Dichlorophen, sulphone, a thiocarbamate and biphenol performed best, killing the full spectrum of isolates cost-effectively. Certain expensive products performed rather poorly, e.g. isothiazoline and MBT. This study highlights the selective action of many bactericides and the inherent resistance of bacteria to a number of different bactericides. This implies the importance of matching bactericides to the dominant bacteria in systems

Cloning and characterization of a 4-Hydroxyphenylacetate 3-Hydroxylase from the Thermophile Geobacillus sp. PA-9

A 4-hydroxyphenylacetic acid (4-HPA) hydroxylase-encoding gene, on a 2.7-kb genomic DNA fragment, was cloned from the thermophile Geobacillus sp. PA-9. The Geobacillus sp. PA-9 4-HPA hydroxylase gene, designated hpaH, encodes a protein of 494 amino acids with a predicted molecular mass of 56.269 Da. The deduced amino-acid sequence of the hpaH gene product displayed <30% amino-acid sequence identity with the larger monooxygenase components of the previously characterized two-component 4-HPA 3-hydroxylases from Escherichia coli W and Klebsiella pneumoniae M5a1. A second oxidoreductase component was not present on the 2.7-kb genomic DNA fragment. The deduced amino-acid sequence of a second C-terminal truncated open reading frame, designated hpaI, exhibited homology to extradiol oxygenases and displayed the highest amino-acid sequence identity (43%) with the 3,4-dihydroxyphenylacetate 2,3-dioxygenase of Arthrobacter globiformis, encoded by mndD. These results, along with catalytic activity observed in crude intracellular extracts prepared from Escherichia coli cells expressing hpaH, is in support of a role for hpaH in the 4-HPA degradative pathway of Geobacillus sp. PA-9.International Foundation for Science, the Council for Development of Social Science Research in Africa and the Makerere University Staff Development Fund

The effect of bactericide treatment on planktonic bacterial communities in water cooling systems

Bactericides were applied to experimental open recirculating cooling-water systems at concentrations found to be effective under laboratory pure-culture conditions. Total aerobic plate counts and bacterial population structures were determined over a period of 48h. In all cases the total aerobic count increased one day after the bactericide addition, and decreased rapidly after ca. 36 to 40h. Population shifts occurred during the course of all four treatments. In all cases different species became dominant concurring with fluctuations in the planktonic plate count, indicating the stress reaction of the biofilm. The species diversity decreased after treatment with dichlorophen, triocarbamate and methylenebis-thiocyanate, and increased upon treatment with humic acid. Species susceptible to bactericides in pure culture were found to be the dominant planktonic survivors. An example is Pseudomonas stutzeri which was the dominant survivor after treatment with thiocarbamate and with diclhlorophen

Effect of storage time and temperature on the aerobic plate count and on the community structure of two water samples

The effect of storage at various temperatures on the bacterial community of a cooling-water sample and a tap-water sample was determined. Samples were stored at 4, 10, 20 and 30 degrees C for 24, 48, 72 and 216 h and the aerobic plate count and bacterial community structure of each were determined using R2A and R3A agars. The culturable count (aerobic plate count on R2A/R3A agar) in both samples varied over time, even after 24 h storage at 4 degrees C, showing that bacterial communities in water are dynamic, even at refrigerator temperatures. At 4 degrees C the culturable count of cooling water initially decreased, followed by a tenfold increase. The tap-water count decreased at 4degrees C. At 10degrees C the pattern was similar. At 20 and 30 degrees C there was a tenfold increase in the culturable count of the cooling water, even after 24 h. In the cooling-water sample, the dominant isolates throughout were Pseudomonas stutzeri and an unidentified Gram negative pink isolate. This isolate was not detected in previous studies where Std I nutrient agar was used. Possibly this isolate plays an important role in cooling-water ecology, but does not grow on the conventional agars. The other isolates appeared randomly on the agar plates. The tap-water sample showed great variation in dominance of species over time. No direct tendencies of rate of decrease or increase could be detected in any of the samples, either in the culturable count or in community structure. Therefor results of analysis after storage cannot be adapted by a pre-determined factor. They must be interpreted with extreme caution, as they do not of necessity reflect the bacterial composition of the sample as drawn, both in terms of total numbers and in terms of community structure. Only counts performed on fresh samples yield reliable results on the total culturable count, and only community structures performed immediately, reflect the state of the community in the system from which the sample was drawn