Extremely halophilic archaeal communities are resilient to short-term entombment in halite.

Some haloarchaea avoid the harsh conditions present in evaporating brines by entombment in brine inclusions within forming halite crystals, where a subset of haloarchaea survives over geological time. However, shifts in the community structure of halite-entombed archaeal communities remain poorly understood. Therefore, we analysed archaeal communities from in situ hypersaline brines collected from Trapani saltern (Sicily) and their successional changes in brines versus laboratory-grown halite over 21 weeks, using high-throughput sequencing. Haloarchaea were dominant, comprising >95% of the archaeal community. Unexpectedly, the OTU richness of the communities after 21 weeks was indistinguishable from the parent brine and overall archaeal abundance in halite showed no clear temporal trends. Furthermore, the duration of entombment was less important than the parent brine from which the halite derived in determining the community composition and relative abundances of most genera in halite-entombed communities. These results show that halite-entombed archaeal communities are resilient to entombment durations of up to 21 weeks, and that entombment in halite may be an effective survival strategy for near complete communities of haloarchaea. Additionally, the dominance of 'halite specialists' observed in ancient halite must occur over periods of years, rather than months, hinting at long-term successional dynamics in this environment.

The ecological impacts of multiple environmental stressors on coastal biofilm bacteria.

Ecological communities are increasingly exposed to multiple interacting stressors. For example, warming directly affects the physiology of organisms, eutrophication stimulates the base of the food web, and harvesting larger organisms for human consumption dampens top-down control. These stressors often combine in the natural environment with unpredictable results. Bacterial communities in coastal ecosystems underpin marine food webs and provide many important ecosystem services (e.g. nutrient cycling and carbon fixation). Yet, how microbial communities will respond to a changing climate remains uncertain. Thus, we used marine mesocosms to examine the impacts of warming, nutrient enrichment, and altered top-predator population size structure (common shore crab) on coastal microbial biofilm communities in a crossed experimental design. Warming increased bacterial α-diversity (18% increase in species richness and 67% increase in evenness), but this was countered by a decrease in α-diversity with nutrient enrichment (14% and 21% decrease for species richness and evenness, respectively). Thus, we show some effects of these stressors could cancel each other out under climate change scenarios. Warming and top-predator population size structure both affected bacterial biofilm community composition, with warming increasing the abundance of bacteria capable of increased mineralization of dissolved and particulate organic matter, such as Flavobacteriia, Sphingobacteriia, and Cytophagia. However, the community shifts observed with warming depended on top-predator population size structure, with Sphingobacteriia increasing with smaller crabs and Cytophagia increasing with larger crabs. These changes could alter the balance between mineralization and carbon sequestration in coastal ecosystems, leading to a positive feedback loop between warming and CO2 production. Our results highlight the potential for warming to disrupt microbial communities and biogeochemical cycling in coastal ecosystems, and the importance of studying these effects in combination with other environmental stressors.

Protein expression in the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 during alkane degradation and cold tolerance.

In cold marine environments, the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC-MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n-alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n-C14 ) at 16°C and 4°C have been quantified. During growth on n-C14 , O. antarctica expressed a complete pathway for the terminal oxidation of n-alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty-acid-CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3- to 21-fold compared with growth on a non-hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species-specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T.

Alcanivorax borkumensis SK2T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C14 ) or branched (pristane) alkanes. During growth on n-C14 , A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for ß-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of ß-oxidation proteins to overcome steric hinderance from branched substrates.

Acclimation of Emiliania huxleyi (1516) to nutrient limitation involves precise modification of the proteome to scavenge alternative sources of N and P.

Limitation of marine primary production by the availability of nitrogen or phosphorus is common. Emiliania huxleyi, a ubiquitous phytoplankter that plays key roles in primary production, calcium carbonate precipitation and production of dimethyl sulfide, often blooms in mid-latitude at the beginning of summer when inorganic nutrient concentrations are low. To understand physiological mechanisms that allow such blooms, we examined how the proteome of E. huxleyi (strain 1516) responds to N and P limitation. We observed modest changes in much of the proteome despite large physiological changes (e.g. cellular biomass, C, N and P) associated with nutrient limitation of growth rate. Acclimation to nutrient limitation did however involve significant increases in the abundance of transporters for ammonium and nitrate under N limitation and for phosphate under P limitation. More notable were large increases in proteins involved in the acquisition of organic forms of N and P, including urea and amino acid/polyamine transporters and numerous C-N hydrolases under N limitation and a large upregulation of alkaline phosphatase under P limitation. This highly targeted reorganization of the proteome towards scavenging organic forms of macronutrients gives unique insight into the molecular mechanisms that underpin how E. huxleyi has found its niche to bloom in surface waters depleted of inorganic nutrients.

amoA Gene abundances and nitrification potential rates suggest that benthic ammonia-oxidizing bacteria and not Archaea dominate N cycling in the Colne Estuary, United Kingdom.

Nitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 µmol N grams dry weight [gdw](-1) day(-1) in June, increasing to 37.4 µmol N gdw(-1) day(-1) in January). At the estuary head, the nitrification potential was 4.3 µmol N gdw(-1) day(-1) in June, increasing to 11.7 µmol N gdw(-1) day(-1) in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOB amoA gene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally. Nitrosomonas spp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOA amoA gene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possibly Nitrosomonas spp.) were of major significance in nitrification.

Impact of a simulated oil spill on benthic phototrophs and nitrogen-fixing bacteria in mudflat mesocosms.

Coastal and estuarine ecosystems are highly susceptible to crude oil pollution. Therefore, in order to examine the resilience of benthic phototrophs that are pivotal to coastal ecosystem functioning, we simulated an oil spill in tidal mesocosms consisting of intact sediment cores from a mudflat at the mouth of the Colne Estuary, UK. At day 21, fluorescence imaging revealed a bloom of cyanobacteria on the surface of oiled sediment cores, and the upper 1.5 cm thick sediment had 7.2 times more cyanobacterial and 1.7 times more diatom rRNA sequences when treated with oil. Photosystem II operating efficiency (Fq'/Fm') was significantly reduced in oiled sediments at day 7, implying that the initial diatom-dominated community was negatively affected by oil, but this was no longer apparent by day 21. Oil addition significantly reduced numbers of the key deposit feeders, and the decreased grazing pressure is likely to be a major factor in the increased abundance of both diatoms and cyanobacteria. By day 5 concentrations of dissolved inorganic nitrogen were significantly lower in oiled mesocosms, likely resulting in the observed increase in nifH-containing, and therefore potentially dinitrogen-fixing, cyanobacteria. Thus, indirect effects of oil, rather than direct inhibition, are primarily responsible for altering the microphytobenthos.

Plasticity in the proteome of Emiliania huxleyi CCMP 1516 to extremes of light is highly targeted.

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 µmol photons m(-2) s(-1) (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature.

The trade-off between the light-harvesting and photoprotective functions of fucoxanthin-chlorophyll proteins dominates light acclimation in Emiliania huxleyi (clone CCMP 1516).

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast. We tested the hypothesis that changes in the light dependencies of photosynthesis, nonphotochemical quenching and PSII photoinactivation arises from changes in the abundances of chloroplast proteins in Emiliania huxleyi strain CCMP 1516 grown at 30 (Low Light; LL) and 1000 (High Light; HL) µmol photons m(-2) s(-1) photon flux densities. Carbon-specific light-saturated gross photosynthesis rates were not significantly different between cells acclimated to LL and HL. Acclimation to LL benefited cells by increasing biomass-specific light absorption and gross photosynthesis rates under low light, whereas acclimation to HL benefited cells by reducing the rate of photoinactivation of PSII under high light. Differences in the relative abundances of proteins assigned to light-harvesting (Lhcf), photoprotection (LI818-like), and the photosystem II (PSII) core complex accompanied differences in photophysiology: specifically, Lhcf:PSII was greater under LL, whereas LI818:PSII was greater in HL. Thus, photoacclimation in E. huxleyi involved a trade-off amongst the characteristics of light absorption and photoprotection, which could be attributed to changes in the abundance and composition of proteins in the light-harvesting antenna of PSII.

Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats.

Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.

Stratified prokaryote network in the oxic-anoxic transition of a deep-sea halocline.

The chemical composition of the Bannock basin has been studied in some detail. We recently showed that unusual microbial populations, including a new division of Archaea (MSBL1), inhabit the NaCl-rich hypersaline brine. High salinities tend to reduce biodiversity, but when brines come into contact with fresher water the natural haloclines formed frequently contain gradients of other chemicals, including permutations of electron donors and acceptors, that may enhance microbial diversity, activity and biogeochemical cycling. Here we report a 2.5-m-thick chemocline with a steep NaCl gradient at 3.3 km within the water column betweeen Bannock anoxic hypersaline brine and overlying sea water. The chemocline supports some of the most biomass-rich and active microbial communities in the deep sea, dominated by Bacteria rather than Archaea, and including four major new divisions of Bacteria. Significantly higher metabolic activities were measured in the chemocline than in the overlying sea water and underlying brine; functional analyses indicate that a range of biological processes is likely to occur in the chemocline. Many prokaryotic taxa, including the phylogenetically new groups, were confined to defined salinities, and collectively formed a diverse, sharply stratified, deep-sea ecosystem with sufficient biomass to potentially contribute to organic geological deposits.

Hydrological properties predict the composition of microbial communities cycling methane and nitrogen in rivers.

Sediment microbial communities drive the biogeochemical cycles that make rivers globally important sources and sinks of carbon (C) and nitrogen (N). The structure of these communities is strongly determined by the local physico-chemical environment. However, we currently lack an understanding of the factors that determine microbial community structures at the catchment scale. Here, we show that the contribution of groundwater to total river flow (quantified as base flow index; BFI) predicts the structure and diversity of the different microbial functional groups that cycle N and C across nine UK rivers, spanning a geological BFI gradient from 0.23 (clay sediment) to 0.95 (chalk gravel sediment). Furthermore, the GC-content (percentage of guanine-cytosine bases in a DNA sequence) and codon-usage bias of ammonia monooxygenase DNA sequences, and the hydrophobicity and net-charge of the corresponding amino acid sequences, were all strongly correlated with BFI, likely reflecting physiological adaptations to different riverbed sediment structure along the BFI gradient. Our results offer an opportunity to overcome the "paradox of scales" that has seen microbial ecologists focus on small- rather than large-scale environmental variables, enabling us to scale-up our understanding of microbial biogeochemistry to the catchment and beyond.

Effects of Dispersants and Biosurfactants on Crude-Oil Biodegradation and Bacterial Community Succession.

This study evaluated the effects of three commercial dispersants (Finasol OSR 52, Slickgone NS, Superdispersant 25) and three biosurfactants (rhamnolipid, trehalolipid, sophorolipid) in crude-oil seawater microcosms. We analysed the crucial early bacterial response (1 and 3 days). In contrast, most analyses miss this key period and instead focus on later time points after oil and dispersant addition. By focusing on the early stage, we show that dispersants and biosurfactants, which reduce the interfacial surface tension of oil and water, significantly increase the abundance of hydrocarbon-degrading bacteria, and the rate of hydrocarbon biodegradation, within 24 h. A succession of obligate hydrocarbonoclastic bacteria (OHCB), driven by metabolite niche partitioning, is demonstrated. Importantly, this succession has revealed how the OHCB Oleispira, hitherto considered to be a psychrophile, can dominate in the early stages of oil-spill response (1 and 3 days), outcompeting all other OHCB, at the relatively high temperature of 16 °C. Additionally, we demonstrate how some dispersants or biosurfactants can select for specific bacterial genera, especially the biosurfactant rhamnolipid, which appears to provide an advantageous compatibility with Pseudomonas, a genus in which some species synthesize rhamnolipid in the presence of hydrocarbons.

Isoprene-Degrading Bacteria from Soils Associated with Tropical Economic Crops and Framework Forest Trees.

Isoprene, a volatile hydrocarbon emitted largely by plants, plays an important role in regulating the climate in diverse ways, such as reacting with free radicals in the atmosphere to produce greenhouse gases and pollutants. Isoprene is both deposited and formed in soil, where it can be consumed by some soil microbes, although much remains to be understood about isoprene consumption in tropical soils. In this study, isoprene-degrading bacteria from soils associated with tropical plants were investigated by cultivation and cultivation-independent approaches. Soil samples were taken from beneath selected framework forest trees and economic crops at different seasons, and isoprene degradation in soil microcosms was measured after 96 h of incubation. Isoprene losses were 4-31% and 15-52% in soils subjected to a lower (7.2 × 105 ppbv) and a higher (7.2 × 106 ppbv) concentration of isoprene, respectively. Sequencing of 16S rRNA genes revealed that bacterial communities in soil varied significantly across plant categories (framework trees versus economic crops) and the presence of isoprene, but not with isoprene concentration or season. Eight isoprene-degrading bacterial strains were isolated from the soils and, among these, four belong to the genera Ochrobactrum, Friedmanniella, Isoptericola and Cellulosimicrobium, which have not been previously shown to degrade isoprene.

Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5.

Naphthenic acids (NAs) are carboxylic acids with the formula (Cn H2n+Z O2 ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Bacterial Community Legacy Effects Following the Agia Zoni II Oil-Spill, Greece.

In September 2017 the Agia Zoni II sank in the Saronic Gulf, Greece, releasing approximately 500 tonnes of heavy fuel oil, contaminating the Salamina and Athens coastlines. Effects of the spill, and remediation efforts, on sediment microbial communities were quantified over the following 7 months. Five days post-spill, the concentration of measured hydrocarbons within surface sediments of contaminated beaches was 1,093-3,773 µg g-1 dry sediment (91% alkanes and 9% polycyclic aromatic hydrocarbons), but measured hydrocarbons decreased rapidly after extensive clean-up operations. Bacterial genera known to contain oil-degrading species increased in abundance, including Alcanivorax, Cycloclasticus, Oleibacter, Oleiphilus, and Thalassolituus, and the species Marinobacter hydrocarbonoclasticus from approximately 0.02 to >32% (collectively) of the total bacterial community. Abundance of genera with known hydrocarbon-degraders then decreased 1 month after clean-up. However, a legacy effect was observed within the bacterial community, whereby Alcanivorax and Cycloclasticus persisted for several months after the oil spill in formerly contaminated sites. This study is the first to evaluate the effect of the Agia Zoni II oil-spill on microbial communities in an oligotrophic sea, where in situ oil-spill studies are rare. The results aid the advancement of post-spill monitoring models, which can predict the capability of environments to naturally attenuate oil.

Differential Protein Expression During Growth on Medium Versus Long-Chain Alkanes in the Obligate Marine Hydrocarbon-Degrading Bacterium Thalassolituus oleivorans MIL-1.

The marine obligate hydrocarbonoclastic bacterium Thalassolituus oleivorans MIL-1 metabolizes a broad range of aliphatic hydrocarbons almost exclusively as carbon and energy sources. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on medium- (n-C14) or long-chain (n-C28) alkanes. During growth on n-C14, T. oleivorans expresses an alkane monooxygenase system involved in terminal oxidation including two alkane 1-monooxygenases, a ferredoxin, a ferredoxin reductase and an aldehyde dehydrogenase. In contrast, during growth on long-chain alkanes (n-C28), T. oleivorans may switch to a subterminal alkane oxidation pathway evidenced by significant upregulation of Baeyer-Villiger monooxygenase and an esterase, proteins catalyzing ketone and ester metabolism, respectively. The metabolite (primary alcohol) generated from terminal oxidation of an alkane was detected during growth on n-C14 but not on n-C28 also suggesting alternative metabolic pathways. Expression of both active and passive transport systems involved in uptake of long-chain alkanes was higher when compared to the non-hydrocarbon control, including a TonB-dependent receptor, a FadL homolog and a specialized porin. Also, an inner membrane transport protein involved in the export of an outer membrane protein was expressed. This study has demonstrated the substrate range of T. oleivorans is larger than previously reported with growth from n-C10 up to n-C32. It has also greatly enhanced our understanding of the fundamental physiology of T. oleivorans, a key bacterium that plays a significant role in natural attenuation of marine oil pollution, by identifying key enzymes expressed during the catabolism of n-alkanes.

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.