Sorption of Cellulases in Biofilm Enhances Cellulose Degradation by Bacillus subtilis.

Biofilm commonly forms on the surfaces of cellulosic biomass but its roles in cellulose degradation remain largely unexplored. We used Bacillus subtilis to study possible mechanisms and the contributions of two major biofilm components, extracellular polysaccharides (EPS) and TasA protein, to submerged biofilm formation on cellulose and its degradation. We found that biofilm produced by B. subtilis is able to absorb exogenous cellulase added to the culture medium and also retain self-produced cellulase within the biofilm matrix. The bacteria that produced more biofilm degraded more cellulose compared to strains that produced less biofilm. Knockout strains that lacked both EPS and TasA formed a smaller amount of submerged biofilm on cellulose than the wild-type strain and also degraded less cellulose. Imaging of biofilm on cellulose suggests that bacteria, cellulose, and cellulases form cellulolytic biofilm complexes that facilitate synergistic cellulose degradation. This study brings additional insight into the important functions of biofilm in cellulose degradation and could potentiate the development of biofilm-based technology to enhance biomass degradation for biofuel production.

Distinguishing Anuran species by high-resolution melting analysis of the COI barcode (COI-HRM).

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High-resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high-resolution melting analysis of COI barcodes (COI-HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.

Rapid differentiation of bacterial communities using high resolution melting analysis.

Analysis of microbial communities is of broad interest in biology and high throughput sequencing is now the preferred method. However, some studies may not need the level of detail high throughput sequencing provides and its cost may limit the number of samples that can be sequenced. High resolution melting analysis (HRM) of 16S rRNA gene variable regions has been proposed as an efficient and low cost method to prioritize samples for sequencing but more specific primers are needed and its efficacy needs to be confirmed. We tested a more specific pair of primers and compared results concerning the structure of microbial communities in tadpole intestine and feces obtained using HRM, denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing performed in parallel. All three methods showed similar conclusions concerning the communities and revealed communities that differed among intestinal regions and feces. The improved HRM method targets a shorter amplicon in the V3 region of the 16S rRNA gene and uses non-degenerate primers, both of which increase the sensitivity of HRM. The HRM approach was shown to be as effective as DGGE for comparing microbial communities, is considerably easier to perform, and can be used to assess differences in microbial community structure among a large number of samples before committing to sequencing.

Complex carbohydrates reduce the frequency of antagonistic interactions among bacteria degrading cellulose and xylan.

Bacterial competition for resources is common in nature but positive interactions among bacteria are also evident. We speculate that the structural complexity of substrate might play a role in mediating bacterial interactions. We tested the hypothesis that the frequency of antagonistic interactions among lignocellulolytic bacteria is reduced when complex polysaccharide is the main carbon source compared to when a simple sugar such as glucose is available. Results using all possible pairwise interactions among 35 bacteria isolated from salt marsh detritus showed that the frequency of antagonistic interactions was significantly lower on carboxymethyl cellulose (CMC)-xylan medium (7.8%) than on glucose medium (15.5%). The two interaction networks were also different in their structures. Although 75 antagonistic interactions occurred on both media, there were 115 that occurred only on glucose and 20 only on CMC-xylan, indicating that some antagonistic interactions were substrate specific. We also found that the frequency of antagonism differed among phylogenetic groups. Gammaproteobacteria and Bacillus sp. were the most antagonistic and they tended to antagonize Bacteroidetes and Actinobacteria, the most susceptible groups. Results from the study suggest that substrate complexity affects how bacteria interact and that bacterial interactions in a community are dynamic as nutrient conditions change.

Synergistic growth in bacteria depends on substrate complexity.

Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (µ). Bacterial synergism also promoted the production of ß-1,4-glucosidase but not the production of cellobiohydrolase or ß-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment.

A faster method to detect norovirus in oysters using probe hybridization to isolate target RNA before RT-PCR.

Human Noroviruses (HuNoVs) are the most frequent cause of outbreaks of acute gastroenteritis following the ingestion of raw or improperly cooked oysters. Although highly sensitive methods to detect HuNoV in oysters using reverse transcriptase-polymerase chain reaction (RT-PCR) are available, rapid methods to process samples for RT-PCR are still needed. The conventional approach is to concentrate the virus first before RNA purification to maximize assay sensitivity, but the procedures used are cumbersome. We developed a new hybridization method that is much faster and more effective compared to existing technology. The procedure includes an initial extraction of total RNA from the digestive diverticula of oysters using TRI Reagent, followed by HuNoV RNA purification using a capture probe and then HuNoV detection by real-time RT-PCR. The detection limit is approximately 100 PCR detection units of HuNoV per sample. Compared to published methods that require an initial virus concentration step before RNA extraction, the new method is much faster to complete. Approximately 3 h are needed to purify HuNoV RNA using the new method compared to at least 8 h using conventional methods. Coupled with real-time RT-PCR, the new method can detect HuNoV in contaminated oysters within 8 h. The effectiveness of the method was demonstrated using live artificially contaminated oysters and wild oysters.

Relationship of human-associated microbial source tracking markers with Enterococci in Gulf of Mexico waters.

Human and ecosystem health can be damaged by fecal contamination of recreational waters. Microbial source tracking (MST) can be used to specifically detect domestic sewage containing human waste, thereby informing both risk assessment and remediation strategies. Previously, an inter-laboratory collaboration developed standardized PCR methods for a bacterial, an archaeal, and a viral indicator of human sewage. Here we present results for two subsequent years of field testing in fresh and salt water by five laboratories across the U.S. Gulf Coast (two in Florida and one each in Mississippi, Louisiana and Texas) using common standard operating procedures (SOPs) developed previously. Culturable enterococci were enumerated by membrane filtration, and PCR was used to detect three MST markers targeting domestic sewage: human-associated Bacteroides (HF183), Methanobrevibacter smithii and human polyomaviruses BK and JC (HPyVs). Detection of sewage markers in surface waters was significantly associated with higher enterococci levels and with exceedance of the recreational water quality standard in four or three regions, respectively. Sewage markers were frequently co-detected in single samples, e.g., M. smithii and HF183 were co-detected in 81% of Louisiana samples, and HPyVs and M. smithii were co-detected in over 40% of southwest Florida and Mississippi samples. This study demonstrates the robustness and inter-laboratory transferability of these three markers for the detection of pollution from domestic sewage in the waters impacting the Gulf of Mexico over a coastal range of over 1000 miles.

Enhancing PCR amplification of DNA from recalcitrant plant specimens using a trehalose-based additive.

PREMISE OF THE STUDY: PCR amplification of DNA extracted from plants is sometimes difficult due to the presence of inhibitory compounds. An effective method to overcome the inhibitory effect of compounds that contaminate DNA from difficult plant specimens is needed. • METHODS AND RESULTS: The effectiveness of a PCR additive reagent containing trehalose, bovine serum albumin (BSA), and polysorbate-20 (Tween-20) (TBT-PAR) was tested. PCR of DNA extracted from fresh, silica-dried, and herbarium leaf material of species of Achariaceae, Asteraceae, Lacistemataceae, and Samydaceae that failed using standard techniques were successful with the addition of TBT-PAR. • CONCLUSIONS: The addition of TBT-PAR during routine PCR is an effective method to improve amplification of DNA extracted from herbarium specimens or plants that are known to contain PCR inhibitors.

A filter-based propidium monoazide technique to distinguish live from membrane-compromised microorganisms using quantitative PCR.

Propidium monoazide (PMA) was used to differentiate live from membrane-compromised bacteria in PCR methods. We have adapted this technique for use on membrane-filtered water samples and determined its efficacy using qPCR. Independent labs at three institutions replicated these findings.

Taura syndrome virus loads in Litopenaeus vannamei hemolymph following infection are related to differential mortality.

Taura syndrome is an economically important disease that can cause catastrophic losses of farmed shrimp. Without effective treatments for Taura syndrome virus (TSV), one approach to managing the problem is to selectively breed shrimp populations with increased disease resistance. To better understand why some shrimp can survive exposure to TSV, information is needed on how viral loads progress and persist following infection. Data reported here show that mortalities occurring mostly within 1 wk of infection are associated with high viral titers, and titers as high as 10(8.7) genome copies per microl hemolymph can persist for up to 3 wk in survivors. Thereafter, and up to approximately 9 wk post-exposure, most surviving shrimp remain chronically infected with TSV loads ranging from 10(4) to 10(8) genome copies per microl hemolymph. Challenging shrimp from families with varying TSV resistance showed that in shrimp from less resistant families, the TSV load in hemolymph increased earlier and reached higher peaks than in shrimp from more resistant families. Although TSV loads in moribund shrimp from families differing in resistance did not differ significantly, infection loads among survivors were lower in shrimp from more resistant families. Taken together, the data suggest that lethal infection loads can occur in both more and less susceptible shrimp and that survivors represent shrimp in which viral expansion is better contained.

Methanobrevibacter ruminantium as an indicator of domesticated-ruminant fecal pollution in surface waters.

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.

Molecular characterization of the insulin-like growth factor-I (IGF-I) gene in channel catfish (Ictalurus punctatus).

The insulin-like growth factor I (IGF-I) gene was characterized in channel catfish. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained in 5'- and 3'-RACE experiments. Direct sequencing of two bacterial artificial chromosome clones revealed gene structure and provided sequence from 640 bp upstream of the initiator methionine to 136 bp beyond the polyadenylation site. Genomic sequence contained a putative TATA box 506 bp upstream of the initiator methionine. The 477-bp reading frame within five exons encoded a 159-amino acid (aa) pre-propeptide highly similar to IGF-I in higher vertebrates. The sequence encoding the signal peptide was unique in catfish and contained 70% G+C content with the potential for a stable stem-loop structure. Full-length cDNA was only maintained in recombination-deficient (DH10B) strain E. coli. Levels of IGF-I mRNA were highest in liver, followed by brain and muscle, then heart and kidney (P<0.05). A CT/GA dinucleotide microsatellite in intron 1 was highly polymorphic in commercial channel catfish, and permitted placement of the IGF-I gene on the catfish genetic map. However, specific IGF-I alleles were not correlated with differences in growth rate from 100 to 130 days post-hatch in USDA103 line catfish.

Methods to increase fidelity of repetitive extragenic palindromic PCR fingerprint-based bacterial source tracking efforts.

The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearson's Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffrey's x, and Ochiai. When enterococcal and Escherichia coli isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this source's isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when E. coli fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.

The use of differential display to isolate viral genomic sequence for rapid development of PCR-based detection methods. A test case using Taura syndrome virus.

The purpose of this study was to explore the efficacy of using differential display (DD) to isolate viral genomic sequence using tissues from infected organisms so that a PCR procedure to detect the pathogen may be developed rapidly. The model virus used was the Taura syndrome virus (TSV), a ssRNA virus that cause high rates of mortality at shrimp farms. Two random primers in combination with four anchored primers were used to isolate five cDNAs, ranging in size from 241 to 822 bp, that were differentially expressed in TSV-infected shrimp (Litopenaeus vannamei). PCR experiments revealed that four of the five encoded shrimp genes while the fifth was likely to be a TSV gene. Evidence that the putative TSV sequence is part of the TSV genome was obtained by the 97% sequence identity it shared with the published TSV genome. PCR primers were designed successfully using the differential display sequence to develop a RT-PCR-based method to detect TSV. Because differential display does not require physical isolation of the virus and only a small amount of infected sample is needed, the technique may be useful as a method to isolate nucleic acid sequences from emerging pathogens so that PCR primers for their detection may be developed rapidly.