Vibrio natriegens genome-scale modeling reveals insights into halophilic adaptations and resource allocation.

Vibrio natriegens is a Gram-negative bacterium with an exceptional growth rate that has the potential to become a standard biotechnological host for laboratory and industrial bioproduction. Despite this burgeoning interest, the current lack of organism-specific qualitative and quantitative computational tools has hampered the community's ability to rationally engineer this bacterium. In this study, we present the first genome-scale metabolic model (GSMM) of V. natriegens. The GSMM (iLC858) was developed using an automated draft assembly and extensive manual curation and was validated by comparing predicted yields, central metabolic fluxes, viable carbon substrates, and essential genes with empirical data. Mass spectrometry-based proteomics data confirmed the translation of at least 76% of the enzyme-encoding genes predicted to be expressed by the model during aerobic growth in a minimal medium. iLC858 was subsequently used to carry out a metabolic comparison between the model organism Escherichia coli and V. natriegens, leading to an analysis of the model architecture of V. natriegens' respiratory and ATP-generating system and the discovery of a role for a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data were further used to investigate additional halophilic adaptations of V. natriegens. Finally, iLC858 was utilized to create a Resource Balance Analysis model to study the allocation of carbon resources. Taken together, the models presented provide useful computational tools to guide metabolic engineering efforts in V. natriegens.

Engineering xylose induction in Vibrio natriegens for biomanufacturing applications.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m2/g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis.IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

A comparison of methods for DNA preparation prior to microarray analysis.

Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase I-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91-100% sensitivity and 100% specificity in the tested strains, requiring half as many manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.

Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production.

A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources ("feedstock flexibility"). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.

Biofilm community structure and the associated drag penalties of a groomed fouling release ship hull coating.

Grooming is a proactive method to keep a ship's hull free of fouling. This approach uses a frequent and gentle wiping of the hull surface to prevent the recruitment of fouling organisms. A study was designed to compare the community composition and the drag associated with biofilms formed on a groomed and ungroomed fouling release coating. The groomed biofilms were dominated by members of the Gammaproteobacteria and Alphaproteobacteria as well the diatoms Navicula, Gomphonemopsis, Cocconeis, and Amphora. Ungroomed biofilms were characterized by Phyllobacteriaceae, Xenococcaceae, Rhodobacteraceae, and the pennate diatoms Cyclophora, Cocconeis, and Amphora. The drag forces associated with a groomed biofilm (0.75 ± 0.09 N) were significantly less than the ungroomed biofilm (1.09 ± 0.06 N). Knowledge gained from this study has helped the design of additional testing which will improve grooming tool design, minimizing the growth of biofilms and thus lowering the frictional drag forces associated with groomed surfaces.

Indole signalling and (micro)algal auxins decrease the virulence of Vibrio campbellii, a major pathogen of aquatic organisms.

Vibrios belonging to the Harveyi clade are major pathogens of marine vertebrates and invertebrates, causing major losses in wild and cultured organisms. Despite their significant impact, the pathogenicity mechanisms of these bacteria are not yet completely understood. In this study, the impact of indole signalling on the virulence of Vibrio campbellii was investigated. Elevated indole levels significantly decreased motility, biofilm formation, exopolysaccharide production and virulence to crustacean hosts. Indole furthermore inhibited the three-channel quorum sensing system of V. campbellii, a regulatory mechanism that is required for full virulence of the pathogen. Further, indole signalling was found to interact with the stress sigma factor RpoS. Together with the observations that energy-consuming processes (motility and bioluminescence) are downregulated, and microarray-based transcriptomics demonstrating that indole decreases the expression of genes involved in energy and amino acid metabolism, the data suggest that indole is a starvation signal in V. campbellii. Finally, it was found that the auxins indole-3-acetic acid and indole-3-acetamide, which were produced by various (micro)algae sharing the aquatic environment with V. campbellii, have a similar effect as observed for indole. Auxins might, therefore, have a significant impact on the interactions between vibrios, (micro)algae and higher organisms, with major ecological and practical implications.

Prevalence of Quinolone Resistance in Enterobacteriaceae from Sierra Leone and the Detection of qnrB Pseudogenes and Modified LexA Binding Sites.

A collection of 74 Enterobacteriaceae isolates found in Bo, Sierra Leone, were tested for quinolone antibiotic susceptibility and resistance mechanisms. The majority of isolates (62%) were resistant to quinolones, and 61% harbored chromosomal gyrA and/or parC mutations. Plasmid-mediated quinolone resistance genes were ubiquitous, with qnrB and aac(6')-Ib-cr being the most prevalent. Mutated LexA binding sites were found in all qnrB1 genes, and truncated qnrB pseudogenes were found in the majority of Citrobacter isolates.

High prevalence of multidrug resistant Enterobacteriaceae isolated from outpatient urine samples but not the hospital environment in Bo, Sierra Leone.

BACKGROUND: The rising level of antimicrobial resistance among bacterial pathogens is one of the most significant public health problems globally. While the antibiotic resistance of clinically important bacteria is closely tracked in many developed countries, the types and levels of resistance and multidrug resistance (MDR) among pathogens currently circulating in most countries of sub-Saharan Africa are virtually unknown. METHODS: From December 2013 to April 2014, we collected 93 urine specimens from all outpatients showing symptoms of urinary tract infection (UTI) and 189 fomite swabs from a small hospital in Bo, Sierra Leone. Culture on chromogenic agar combined with biochemical and DNA sequence-based assays was used to detect and identify the bacterial isolates. Their antimicrobial susceptibilities were determined using a panel of 11 antibiotics or antibiotic combinations. RESULTS: The 70 Enterobacteriaceae urine isolates were identified as Citrobacter freundii (n = 22), Klebsiella pneumoniae (n = 15), Enterobacter cloacae (n = 15), Escherichia coli (n = 13), Enterobacter sp./Leclercia sp. (n = 4) and Escherichia hermannii (n = 1). Antimicrobial susceptibility testing demonstrated that 85.7 % of these isolates were MDR while 64.3 % produced an extended-spectrum ß-lactamase (ESBL). The most notable observations included widespread resistance to sulphonamides (91.4 %), chloramphenicol (72.9 %), gentamycin (72.9 %), ampicillin with sulbactam (51.4 %) and ciprofloxacin (47.1 %) with C. freundii exhibiting the highest and E. coli the lowest prevalence of multidrug resistance. The environmental cultures resulted in only five Enterobacteriaceae isolates out of 189 collected with lower overall antibiotic resistance. CONCLUSIONS: The surprisingly high proportion of C. freundii found in urine of patients with suspected UTI supports earlier findings of the growing role of this pathogen in UTIs in low-resource countries. The isolates of all analyzed species showed worryingly high levels of resistance to both first- and second-line antibiotics as well as a high frequency of MDR and ESBL phenotypes, which likely resulted from the lack of consistent antibiotic stewardship policies in Sierra Leone. Analysis of hospital environmental isolates however suggested that fomites in this naturally ventilated hospital were not a major reservoir for Enterobacteriaceae or antibiotic resistance determinants.

Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome.

Microorganisms that respire electrodes may be exploited for biotechnology applications if key pathways for extracellular electron transfer can be identified and manipulated through bioengineering. To determine whether expression of proposed Biocathode-MCL extracellular electron transfer proteins are changed by modulating electrode potential without disrupting the relative distribution of microbial constituents, metaproteomic and 16S rRNA gene expression analyses were performed after switching from an optimal to suboptimal potential based on an expected decrease in electrode respiration. Five hundred and seventy-nine unique proteins were identified across both potentials, the majority of which were assigned to three previously defined Biocathode-MCL metagenomic clusters: a Marinobacter sp., a member of the family Chromatiaceae, and a Labrenzia sp (abbreviated as MCL). Statistical analysis of spectral counts using the Fisher's exact test identified 16 proteins associated with the optimal potential, five of which are predicted electron transfer proteins. The majority of proteins associated with the suboptimal potential were involved in protein turnover/synthesis, motility, and membrane transport. Unipept and 16S rRNA gene expression analyses indicated that the taxonomic profile of the microbiome did not change after 52 h at the suboptimal potential. These findings show that protein expression is sensitive to the electrode potential without inducing shifts in community composition, a feature that may be exploited for engineering Biocathode-MCL. All MS data have been deposited in the ProteomeXchange with identifier PXD001590 (http://proteomecentral.proteomexchange.org/dataset/PXD001590).

Unique and conserved genome regions in Vibrio harveyi and related species in comparison with the shrimp pathogen Vibrio harveyi CAIM 1792.

Vibrio harveyi CAIM 1792 is a marine bacterial strain that causes mortality in farmed shrimp in north-west Mexico, and the identification of virulence genes in this strain is important for understanding its pathogenicity. The aim of this work was to compare the V. harveyi CAIM 1792 genome with related genome sequences to determine their phylogenic relationship and explore unique regions in silico that differentiate this strain from other V. harveyi strains. Twenty-one newly sequenced genomes were compared in silico against the CAIM 1792 genome at nucleotidic and predicted proteome levels. The proteome of CAIM 1792 had higher similarity to those of other V. harveyi strains (78%) than to those of the other closely related species Vibrio owensii (67%), Vibrio rotiferianus (63%) and Vibrio campbellii (59%). Pan-genome ORFans trees showed the best fit with the accepted phylogeny based on DNA-DNA hybridization and multi-locus sequence analysis of 11 concatenated housekeeping genes. SNP analysis clustered 34/38 genomes within their accepted species. The pangenomic and SNP trees showed that V. harveyi is the most conserved of the four species studied and V. campbellii may be divided into at least three subspecies, supported by intergenomic distance analysis. blastp atlases were created to identify unique regions among the genomes most related to V. harveyi CAIM 1792; these regions included genes encoding glycosyltransferases, specific type restriction modification systems and a transcriptional regulator, LysR, reported to be involved in virulence, metabolism, quorum sensing and motility.

A previously uncharacterized, nonphotosynthetic member of the Chromatiaceae is the primary CO2-fixing constituent in a self-regenerating biocathode.

Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (+310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O2, and presumably fixing CO2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha- and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO2 fixation mechanisms for a self-regenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Reprint of "Which metaproteome? The impact of protein extraction bias on metaproteomic analyses".

Culture-independent techniques such as LC-MS/MS-based metaproteomic analyses are being increasingly utilized for the study of microbial composition and function in complex environmental samples. Although several studies have documented the many challenges and sources of bias that must be considered in these types of analyses, none have systematically characterized the effect of protein extraction bias on the biological interpretation of true environmental biofilm metaproteomes. In this study, we compared three protein extraction methods commonly used in the analyses of environmental samples [guanidine hydrochloride (GuHCl), B-PER, sequential citrate-phenol (SCP)] using nano-LC-MS/MS and an environmental marine biofilm to determine the unique biases introduced by each method and their effect on the interpretation of the derived metaproteomes. While the protein extraction efficiencies of the three methods ranged from 2.0 to 4.3%, there was little overlap in the sequence (1.9%), function (8.3% of total assigned protein families) and origin of the identified proteins from each extract. Each extraction method enriched for different protein families (GuHCl--photosynthesis, carbohydrate metabolism; B-PER--membrane transport, oxidative stress; SCP--calcium binding, structural) while 23.7-45.4% of the identified proteins lacked SwissProt annotations. Taken together, the results demonstrated that even the most basic interpretations of this complex microbial assemblage (species composition, ratio of prokaryotic to eukaryotic proteins, predominant functions) varied with little overlap based on the protein extraction method employed. These findings demonstrate the heavy influence of protein extraction on biofilm metaproteomics and provide caveats for the interpretation of such data sets when utilizing single protein extraction methods for the description of complex microbial assemblages.

Integrated metagenomic and metaproteomic analyses of marine biofilm communities.

Metagenomic and metaproteomic analyses were utilized to determine the composition and function of complex air-water interface biofilms sampled from the hulls of two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S rDNA profiling revealed two significantly different and taxonomically rich biofilm communities (6,942 taxa) in which the majority of unique taxa were ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and Clostridia. Although metagenomic sequencing indicated that both biofilms were dominated by prokaryotic sequence reads (> 91%) with the majority of the bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome harbored greater organismal and functional diversity and was comparatively enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid chromatography-tandem mass spectrometry metaproteome analyses identified 678 unique proteins, revealed little overlap in species and protein composition between the ships and contrasted with the metagenomic data in that ~80% of classified and annotated proteins were of eukaryotic origin and dominated by members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome, quantitative (18)O and iTRAQ analyses demonstrated a significantly greater abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom photosynthesis proteins on Ship-2. Photosynthetic pigment composition and elemental analyses confirmed that both biofilms were dominated by phototrophic processes. These data begin to provide a better understanding of the complex organismal and biomolecular composition of marine biofilms while highlighting caveats in the interpretation of stand-alone environmental '-omics' datasets.

Impact of template denaturation prior to whole genome amplification on gene detection in high GC-content species, Burkholderia mallei and B. pseudomallei.

OBJECTIVE: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM). RESULTS: Whole genome amplicons from 22 B. mallei (BM) and 37 B. pseudomallei (BP) isolates were tested for > 500 ARDs using ARDM v.3.1. ARDM detected the following Burkholderia spp.-derived genes, aac(6), blaBP/MBL-3, blaABPS, penA-BP, and qacE, in both BM and BP while blaBP/MBL-1, macB, blaOXA-42/43 and penA-BC were observed in BP only. The method of denaturing template for whole genome amplification greatly affected the numbers and types of genes detected by the ARDM. BlaTEM was detected in nearly a third of BM and BP amplicons derived from thermally, but not chemically denatured templates. BlaTEM results were confirmed by PCR, with 81% concordance between methods. Sequences from 414-nt PCR amplicons (13 preparations) were 100% identical to the Klebsiella pneumoniae reference gene. Although blaTEM sequences have been observed in B. glumae, B. cepacia, and other undefined Burkholderia strains, this is the first report of such sequences in BM/BP/B. thailandensis (BT) clade. These results highlight the importance of sample preparation in achieving adequate genome coverage in methods requiring untargeted amplification before analysis.