Uncovering potential interspecies signaling factors in plant-derived mixed microbial culture.

Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.

Effects of sample preservation on marine microbial diversity analysis.

Three replicate seawater samples were collected on three different days, filtered immediately and preserved with one of two guanidinium thiocyanate-based preservatives (DNAzol™ or RNA Lysis Buffer™ plus ß-mercaptoethanol (RLA+)) and were kept frozen while being shipped to a lab. In parallel, a carboy of seawater was collected on each of the three days and maintained at ambient temperature while being shipped to a lab. Upon receipt the samples were filtered and treated in the same manner as for immediate preservation. Significantly more DNA was obtained from samples immediately preserved with DNAzol than the corresponding shipped samples for 2 of the 3 days. More DNA was extracted from DNAzol preserved samples but more RNA was obtained from RLA+ preserved samples. A protocol was designed to extract both DNA and RNA from split samples preserved with RLA+ and cDNA was synthesized from the RNA. Three high-throughput 16S rRNA gene libraries were constructed, one from DNA preserved with DNAzol, one from DNA preserved with RLA+ and one from cDNA (RLA+ preserved). Greater alpha diversity was found for libraries constructed from immediately preserved vs. shipped samples for both preservation types, with immediate preservation with DNAzol obtaining the highest level of diversity. Libraries constructed from immediately preserved (RLA+) DNA had greater alpha diversity than libraries constructed from shipped preserved (RLA+) DNA or cDNA. Unifrac measures of beta diversity showed clearer separation of sample types and a greater % variance explained for weighted than for unweighted principal coordinate analysis (PCoA) plots, indicating sample types varied more in their relative abundance of taxa than the presence/absence of particular taxa. We recommend immediate preservation of seawater samples, with DNAzol as the preferred preservative if quantification via qPCR will be performed or the highest alpha diversity is desired but preservation with RLA+ if RNA will be extracted.

Metabolomic and Metagenomic Analysis of Two Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion.

Corrosion processes in two North Sea oil production pipelines were studied by analyzing pig envelope samples via metagenomic and metabolomic techniques. Both production systems have similar physico-chemical properties and injection waters are treated with nitrate, but one pipeline experiences severe corrosion and the other does not. Early and late pigging material was collected to gain insight into the potential causes for differential corrosion rates. Metabolites were extracted and analyzed via ultra-high performance liquid chromatography/high-resolution mass spectrometry with electrospray ionization (ESI) in both positive and negative ion modes. Metabolites were analyzed by comparison with standards indicative of aerobic and anaerobic hydrocarbon metabolism and by comparison to predicted masses for KEGG metabolites. Microbial community structure was analyzed via 16S rRNA gene qPCR, sequencing of 16S PCR products, and MySeq Illumina shotgun sequencing of community DNA. Metagenomic data were used to reconstruct the full length 16S rRNA genes and genomes of dominant microorganisms. Sequence data were also interrogated via KEGG annotation and for the presence of genes related to terminal electron accepting (TEA) processes as well as aerobic and anaerobic hydrocarbon degradation. Significant and distinct differences were observed when comparing the 'high corrosion' (HC) and the 'low corrosion' (LC) pipeline systems, especially with respect to the TEA utilization potential. The HC samples were dominated by sulfate-reducing bacteria (SRB) and archaea known for their ability to utilize simple carbon substrates, whereas LC samples were dominated by pseudomonads with the genetic potential for denitrification and aerobic hydrocarbon degradation. The frequency of aerobic hydrocarbon degradation genes was low in the HC system, and anaerobic hydrocarbon degradation genes were not detected in either pipeline. This is in contrast with metabolite analysis, which demonstrated the presence of several succinic acids in HC samples that are diagnostic of anaerobic hydrocarbon metabolism. Identifiable aerobic metabolites were confined to the LC samples, consistent with the metagenomic data. Overall, these data suggest that corrosion management might benefit from a more refined understanding of microbial community resilience in the face of disturbances such as nitrate treatment or pigging, which frequently prove insufficient to alter community structure toward a stable, less-corrosive assemblage.

Molecular methods resolve the bacterial composition of natural marine biofilms on galvanically coupled stainless steel cathodes.

Navy vessels consist of various metal alloys and biofilm accumulation at the metal surface is thought to play a role in influencing metal deterioration. To develop better strategies to monitor and control metallic biofilms, it is necessary to resolve the bacterial composition within the biofilm. This study aimed to determine if differences in electrochemical current could influence the composition of dominant bacteria in a metallic biofilm, and if so, determine the level of resolution using metagenomic amplicon sequencing. Current was generated by creating galvanic couples between cathodes made from stainless steel and anodes made from carbon steel, aluminum, or copper nickel and exposing them in the Delaware Bay. Stainless steel cathodes (SSCs) coupled to aluminum or carbon steel generated a higher mean current (0.39 mA) than that coupled to copper nickel (0.17 mA). Following 3 months of exposure, the bacterial composition of biofilms collected from the SSCs was determined and compared. Dominant bacterial taxa from the two higher current SSCs were different from that of the low-current SSC as determined by DGGE and verified by Illumina DNA-seq analysis. These results demonstrate that electrochemical current could influence the composition of dominant bacteria in metallic biofilms and that amplicon sequencing is sufficient to complement current methods used to study metallic biofilms in marine environments.

Metabolomic and high-throughput sequencing analysis-modern approach for the assessment of biodeterioration of materials from historic buildings.

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II-Birkenau concentration and extermination camp in Oswiecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial communities and to shed new light on biodeterioration processes.

Issues for storing plant-based alternative fuels in marine environments.

Two coastal seawaters (Key West, FL, USA and the Persian Gulf, Bahrain, representing oligotrophic and eutrophic environments, respectively) were used to evaluate potential biodegradation and corrosion problems during exposure to alternative and conventional fuels. Uncoated carbon steel was exposed at the fuel/seawater interface and polarization resistance was monitored. Under typical marine storage conditions, dioxygen in natural seawater exposed to fuel and carbon steel was reduced to <0.1parts-per-million within 2d due to consumption by corrosion reactions and aerobic microbial respiration. Sulfides, produced by anaerobic sulfate-reducing bacteria, and chlorides were co-located in corrosion products. Transient dioxygen influenced both metabolic degradation pathways and resulting metabolites. Catechols, indicative of aerobic biodegradation, persisted after 90d exposures. Detection of catechols suggested that initial exposure to dioxygen resulted in the formation of aerobic metabolites that exacerbated subsequent corrosion processes.

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

Molecular tools to track bacteria responsible for fuel deterioration and microbiologically influenced corrosion.

Investigating the susceptibility of various fuels to anaerobic biodegradation has become complicated with the recognition that the fuels themselves are not sterile. Bacterial DNA could be obtained when various fuels were filtered through a hydrophobic teflon (0.22 µm) membrane filter. Bacterial 16S rRNA genes from these preparations were PCR amplified, cloned, and the resulting libraries sequenced to identify the fuel-borne bacterial communities. The most common sequence, found in algal- and camelina-based biofuels as well as in ultra-low sulfur diesel (ULSD) and F76 diesel, was similar to that of a Tumebacillus. The next most common sequence was similar to Methylobacterium and was found in the biofuels and ULSD. Higher level phylogenetic groups included representatives of the Firmicutes (Bacillus, Lactobacillus and Streptococcus), several Actinobacteria, Deinococcus-Thermus, Chloroflexi, Cyanobacteria, Bacteroidetes, Alphaproteobacteria (Methylobacterium and Sphingomonadales), Betaproteobacteria (Oxalobacteraceae and Burkholderiales) and Deltaproteobacteria. All of the fuel-associated bacterial sequences, except those obtained from a few facultative microorganisms, were from aerobes and only remotely affiliated with sequences that resulted from anaerobic successional events evident when ULSD was incubated with a coastal seawater and sediment inoculum. Thus, both traditional and alternate fuel formulations harbor a characteristic microflora, but these microorganisms contributed little to the successional patterns that ultimately resulted in fuel decomposition, sulfide formation and metal biocorrosion. The findings illustrate the value of molecular approaches to track the fate of bacteria that might come in contact with fuels and potentially contribute to corrosion problems throughout the energy value chain.

Sulphide production and corrosion in seawaters during exposure to FAME diesel.

Experiments were designed to evaluate the corrosion-related consequences of storing/transporting fatty acid methyl ester (FAME) alternative diesel fuel in contact with natural seawater. Coastal Key West, FL (KW), and Persian Gulf (PG) seawaters, representing an oligotrophic and a more organic- and inorganic mineral-rich environment, respectively, were used in 60 day incubations with unprotected carbon steel. The original microflora of the two seawaters were similar with respect to major taxonomic groups but with markedly different species. After exposure to FAME diesel, the microflora of the waters changed substantially, with Clostridiales (Firmicutes) becoming dominant in both. Despite low numbers of sulphate-reducing bacteria in the original waters and after FAME diesel exposure, sulphide levels and corrosion increased markedly due to microbial sulphide production. Corrosion morphology was in the form of isolated pits surrounded by an intact, passive surface with the deepest pits associated with the fuel/seawater interface in the KW exposure. In the presence of FAME diesel, the highest corrosion rates measured by linear polarization occurred in the KW exposure correlating with significantly higher concentrations of sulphur and chlorine (presumed sulphide and chloride, respectively) in the corrosion products.

Similar gene estimates from circular and linear standards in quantitative PCR analyses using the prokaryotic 16S rRNA gene as a model.

Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples. A recent publication showed that circular plasmid DNA standards grossly overestimated numbers of a target gene by as much as 8-fold in a eukaryotic system using quantitative PCR (qPCR) analysis. Overestimation of microbial numbers is a serious concern in industrial settings where qPCR estimates form the basis for quality control or mitigation decisions. Unlike eukaryotes, bacteria and archaea most commonly have circular genomes and plasmids and therefore may not be subject to the same levels of overestimation. Therefore, the feasibility of using circular DNA plasmids as standards for 16S rRNA gene estimates was assayed using these two prokaryotic systems, with the practical advantage being rapid standard preparation for ongoing qPCR analyses. Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively. Estimates of 16S rRNA gene copies were made based on circular and linearized DNA conformations using two genomes from each domain: Desulfovibrio vulgaris, Pseudomonas aeruginosa, Archaeoglobus fulgidus, and Methanocaldocococcus jannaschii. The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.

The transcription factor Bright plays a role in marginal zone B lymphocyte development and autoantibody production.

Previous data suggested that constitutive expression of the transcription factor Bright (B cell regulator of immunoglobulin heavy chain transcription), normally tightly regulated during B cell differentiation, was associated with autoantibody production. Here we show that constitutive Bright expression results in skewing of mature B lineage subpopulations toward marginal zone cells at the expense of the follicular subpopulation. C57Bl/6 transgenic mice constitutively expressing Bright in B lineage cells generated autoantibodies that were not the result of global increases in immunoglobulin or of breaches in key tolerance checkpoints typically defective in other autoimmune mouse models. Rather, autoimmunity correlated with increased numbers of marginal zone B cells and alterations in the phenotype and gene expression profiles of lymphocytes within the follicular B cell compartment. These data suggest a novel role for Bright in the normal development of mature B cell subsets and in autoantibody production.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Transgenic mice expressing dominant-negative bright exhibit defects in B1 B cells.

The transcription factor Bright up-regulates Ig H chain production from select V region promoters and requires Bright dimerization, Bruton's tyrosine kinase (Btk), and the Btk substrate, TFII-I, for this activity. Defects in Btk cause X-linked immunodeficiency disease in mice and humans. Btk-deficient mice exhibit decreased serum IgM production, B cell developmental blocks, absence of peritoneal B1 cells, and subnormal immune responses against Ags, including phosphorylcholine, which confer protection against Streptococcus pneumoniae. Transgenic mice expressing dominant-negative Bright share similarities with Btk-deficient mice, including decreased serum IgM, poor anti-phosphorylcholine responses, and slightly reduced numbers of mature B cells. Although dominant-negative Bright mice developed B1 B cells, these were functionally deficient in Ig secretion. These data suggest a mechanistic explanation for the abnormal responses to phosphorylcholine observed in Btk-deficient mice, and indicate that Bright functions in a subset of Btk-dependent pathways in vivo, particularly those responses dominated by B1 B cells.

Plesiomonas shigelloides hugZ encodes an iron-regulated heme binding protein required for heme iron utilization.

Plesiomonas shigelloides is an intestinal pathogen that uses heme as an iron source. The P. shigelloides heme utilization system consists of 10 genes, 7 of which permit heme transport and 3 of which are associated with utilization of heme as an iron source once it is inside the cell. The goal of this study was to examine hugZ, 1 of the 3 genes associated with utilization of heme iron. DPH8, a hugZ mutant, failed to grow to full cell density in media containing heme as the iron source, indicating that hugZ is required for heme iron utilization. Western blots using antibodies against Vibrio cholerae HutZ to detect the P. shigelloides HugZ indicated that hugZ encodes an iron-regulated cytoplasmic protein, which is absent in DPH8. A heme affinity bead assay performed on soluble protein fractions from P. shigelloides DPH8/pHUG24.5 (pHUG24.5 encodes hugZ) indicated that HugZ binds heme. Heme utilization was restored in DPH8 by hox1, which encodes the alpha-heme oxygenase from Synechocystis sp. strain PCC6803. However, HugZ did not exhibit alpha-heme oxygenase activity in an assay that detects the conversion of heme to the bilin functional group present in phycobiliproteins. These results do not rule out that HugZ exhibits another type of heme oxygenase activity not detected in the assay.