Draft genome of the fungicidal biological control agent Burkholderia anthina strain XXVI.

Burkholderia anthina XXVI is a rhizosphere bacterium isolated from a mango orchard in Mexico. This strain has a significant biological control activity against the causal agent of mango anthracnose, Colletotrichum gloeosporioides, likely through the production of siderophores and other secondary metabolites. Here, we present a draft genome sequence of B. anthina XXVI (approximately 7.7 Mb; and G + C content of 67.0%), with the aim of gaining insight into the genomic basis of antifungal modes of action, ecological success as a biological control agent, and full biosynthetic potential.

Implications of direct amplification for measuring antimicrobial resistance using point-of-care devices.

Antimicrobial resistance (AMR) is recognized as a global threat to human health. Rapid detection and characterization of AMR is a critical component of most antibiotic stewardship programs. Methods based on amplification of nucleic acids for detection of AMR are generally faster than culture-based approaches but they still require several hours to more than a day due to the need for transporting the sample to a centralized laboratory, processing of sample, and sometimes DNA purification and concentration. Nucleic acids-based point-of-care (POC) devices are capable of rapidly diagnosing antibiotic-resistant infections which may help in making timely and correct treatment decisions. However, for most POC platforms, sample processing for nucleic acids extraction and purification is also generally required prior to amplification. Direct amplification, an emerging possibility for a number of polymerases, has the potential to eliminate these steps without significantly impacting diagnostic performance. This review summarizes direct amplification methods and their implication for rapid measurement of AMR. Future research directions that may further strengthen the possibility of integrating direct amplification methods with POC devices are also summarized.

Coselection of cadmium and benzalkonium chloride resistance in conjugative transfers from nonpathogenic Listeria spp. to other Listeriae.

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.

Impact of space, time and complex environments on microbial communities.

The unparalleled accumulation of biological and contextual data is currently revolutionizing the way environmental microbiologists address ecological questions. Here, we briefly review the likely causes that may explain this remarkable scientific revolution and present a synthesized view about how to describe microbial communities in their complex environmental context.

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/.

Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology.

Antarctic permafrost soils have not received as much geocryological and biological study as has been devoted to the ice sheet, though the permafrost is more stable and older and inhabited by more microbes. This makes these soils potentially more informative and a more significant microbial repository than ice sheets. Due to the stability of the subsurface physicochemical regime, Antarctic permafrost is not an extreme environment but a balanced natural one. Up to 10(4) viable cells/g, whose age presumably corresponds to the longevity of the permanently frozen state of the sediments, have been isolated from Antarctic permafrost. Along with the microbes, metabolic by-products are preserved. This presumed natural cryopreservation makes it possible to observe what may be the oldest microbial communities on Earth. Here, we describe the Antarctic permafrost habitat and biodiversity and provide a model for martian ecosystems.

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data.

Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website (http://rdp.cme.msu.edu/).

Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400.

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the DeltabenABCD::kan mutant), the boxC pathway (the DeltaboxABC::kan mutant), and both pathways (the DeltabenABCDDelta boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the DeltabenABCD::kan and DeltabenABCD DeltaboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the DeltabenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400.

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.

Global transcriptome analysis of Shewanella oneidensis MR-1 exposed to different terminal electron acceptors.

To gain insight into the complex structure of the energy-generating networks in the dissimilatory metal reducer Shewanella oneidensis MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and non-metal electron acceptors. Gene expression patterns were similar irrespective of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate-respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors and PAS-domain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA that were previously linked to the reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual non-metal electron acceptors resulting in the identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multi-cytochrome gene clusters. Collectively, the gene expression results reflect the fundamental differences between metal and non-metal respiratory pathways of S. oneidensis MR-1, where the coordinate induction of detoxification and stress response genes play a key role in adaptation of this organism under metal-reducing conditions. Moreover, the relative paucity and/or the constitutive nature of genes involved in electron transfer to metals is likely due to the low-specificity and the opportunistic nature of the metal-reducing electron transport pathways.

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis.

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101,632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR.

We developed a quantitative competitive PCR (QC-PCR) system to detect and quantify copper-denitrifying bacteria in environmental samples. The primers were specific to copper-dependent nitrite reductase gene (nirK). We were able to detect about 200 copeis of nirK in the presence of abundant non-specific target DNA and about 1.2 x 10(3)Pseudomonas sp. G-179 cells from one gram of sterilized soil by PCR amplification. A 312-bp nirK internal standard (IS) was constructed, which showed very similar amplification efficiency with the target nirKfragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The accuracy of this system was evaluated by quantifying various known amount of nirK DNA. The linear regressions were obtained with a R(2) of 0.9867 for 10(3)copies of nirK, 0.9917 for 10(4) copies of nirK, 0.9899 for 10(5) copies of nirK and 0.9846 for 10(6) copies of nirK. A high correlation between measured nirK and calculated nirK (slope of 1.0398, R(2)=0.9992) demonstrated that an accurate measurement could be achieved with this system. Using this method, we quantified nirK in several A-horizon and stream sediment samples from eastern Tennessee. In general, the abundance of nirK was in the range of 10(8)-10(9) copies g soil(-1) dry weight. The nirK content in the soil samples appeared correlated with NH(4)(N) content in the soil. The activities of copper-denitrifying bacteria were evaluated by quantifying cDNA of nirK. In most of sample examined, the content of nirK cDNA was less than 10(5) copies g soil(-1) dry weight. Higher nirK cDNA content (>10(6) copies g soil(-1) dry weight) was detected from both sediment samples at Rattlebox Creek and the Walker Branch West Ridge. Although the stream sediment samples at the Walker Branch West Ridge contained less half of the nirK gene content as compared to A-horizon sample, the activities of copper-denitrifying bacteria were almost 600 times higher than in the A-horizon sample.

Biphenyl and benzoate metabolism in a genomic context: outlining genome-wide metabolic networks in Burkholderia xenovorans LB400.

We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was up-regulated in benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C(1) metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.

Competitive and interactions affecting a fermentative spirochete in anaerobic chemostats.

Terminal restriction fragment length polymorphism and fluorescent in situ hybridization revealed that spirochete-related populations dominated two glucose-fed methanogenic bioreactor communities at dilution rates of 0.06, 0.13, and 0.17 day(-1). At dilution rates of 0.25 and 0.50 day(-1), spirochete-related populations decreased while Clostridium-related populations increased. Isolates representing both dominant populations were obtained (Treponema R8 and Clostridium S9) and competed against each other in continuous culture. Treponema R8 out-competed Clostridium S9 at all dilution rates applied (0.17 to 1.0 day(-1)) when sufficient pantothenate was supplied in the medium. Without sufficient pantothenate, the population size of Treponema R8 was limited to 40% of the total cells. Coculture of Treponema R8 with Methanobacterium bryantii increased the cell yield of Treponema R8 and relieved the pantothenate requirement. Triculture of Treponema R8, Clostridium S9, and M. bryantii in pantothenate-deficient medium allowed Treponema R8 to outcompete Clostridium S9 in continuous culture upto a dilution rate of 0.50 day(-1). These experiments demonstrate that cofactor and vitamin requirements can affect the competitive success of a microbial species.

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy.

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

A two-species test of the hypothesis that spatial isolation influences microbial diversity in soil.

The hypothesis that spatial isolation is a key determinant of microbial community structure in soils was evaluated by examining the competitive dynamics of two species growing on a single resource in a uniform sand matrix under varied moisture content. One species dominated the community under highly connected, saturated treatments, suggesting that these conditions allow competitive interactions to structure the community. As moisture content decreased, however, the less competitive species became established in the community. This effect was most pronounced at a matric water potential of -0.14 MPa where estimates of final population density and species fitness were equal. A second but more closely related species pair exhibited a similar response to decreasing moisture, suggesting that the effects of spatial isolation we observed are not simply a species-pair-specific phenomenon. These findings indicate that spatial isolation, created by low moisture content, plays an important role in structuring soil microbial communities.

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Simultaneous recovery of RNA and DNA from soils and sediments.

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.

The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions.

We compared carbon flow under constant low-substrate conditions (below 20 microM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring 14C-labeled metabolites derived from a feeding of [U-14C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.