Genomic Aromatic Compound Degradation Potential of Novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov.

We performed a taxonomic and comparative genomics analysis of 67 novel Paraburkholderia isolates from forest soil. Phylogenetic analysis of the recA gene revealed that these isolates formed a coherent lineage within the genus Paraburkholderia that also included Paraburkholderiaaspalathi, Paraburkholderiamadseniana, Paraburkholderiasediminicola, Paraburkholderiacaffeinilytica, Paraburkholderiasolitsugae and Paraburkholderiaelongata and four unidentified soil isolates from earlier studies. A phylogenomic analysis, along with orthoANIu and digital DNA-DNA hybridization calculations revealed that they represented four different species including three novel species and P. aspalathi. Functional genome annotation of the strains revealed several pathways for aromatic compound degradation and the presence of mono- and dioxygenases involved in the degradation of the lignin-derived compounds ferulic acid and p-coumaric acid. This co-occurrence of multiple Paraburkholderia strains and species with the capacity to degrade aromatic compounds in pristine forest soil is likely caused by the abundant presence of aromatic compounds in decomposing plant litter and may highlight a diversity in micro-habitats or be indicative of synergistic relationships. We propose to classify the isolates representing novel species as Paraburkholderia domus with LMG 31832T (=CECT 30334) as the type strain, Paraburkholderia nemoris with LMG 31836T (=CECT 30335) as the type strain and Paraburkholderia haematera with LMG 31837T (=CECT 30336) as the type strain and provide an emended description of Paraburkholderia sediminicola Lim et al. 2008.

Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout.

Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

Genome-based classification of Burkholderia cepacia complex provides new insight into its taxonomic status.

BACKGROUND: Accurate classification of different Burkholderia cepacia complex (BCC) species is essential for therapy, prognosis assessment and research. The taxonomic status of BCC remains problematic and an improved knowledge about the classification of BCC is in particular needed. METHODS: We compared phylogenetic trees of BCC based on 16S rRNA, recA, hisA and MLSA (multilocus sequence analysis). Using the available whole genome sequences of BCC, we inferred a species tree based on estimated single-copy orthologous genes and demarcated species of BCC using dDDH/ANI clustering. RESULTS: We showed that 16S rRNA, recA, hisA and MLSA have limited resolutions in the taxonomic study of closely related bacteria such as BCC. Our estimated species tree and dDDH/ANI clustering clearly separated 116 BCC strains into 36 clusters. With the appropriate reclassification of misidentified strains, these clusters corresponded to 22 known species as well as 14 putative novel species. CONCLUSIONS: This is the first large-scale and systematic study of the taxonomic status of the BCC and could contribute to further insights into BCC taxonomy. Our study suggested that conjunctive use of core phylogeny based on single-copy orthologous genes, as well as pangenome-based dDDH/ANI clustering would provide a preferable framework for demarcating closely related species. REVIEWER: This article was reviewed by Dr. Xianwen Ren.

Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response.

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

TIRF-Based Single-Molecule Detection of the RecA Presynaptic Filament Dynamics.

RecA is a key protein in homologous DNA repair process. On a single-stranded (ss) DNA, which appears as an intermediate structure at a double-strand break site, RecA forms a kilobase-long presynaptic filament that mediates homology search and strand exchange reaction. RecA requires adenosine triphosphate as a cofactor that confers dynamic features to the filament such as nucleation, end-dependent growth and disassembly, scaffold shift along the ssDNA, and conformational change. Due to the complexity of the dynamics, detailed molecular mechanisms of functioning presynaptic filament have been characterized only recently after the advent of single-molecule techniques that allowed real-time observation of each kinetic process. In this chapter, single-molecule fluorescence resonance energy transfer assays, which revealed detailed molecular pictures of the presynaptic filament dynamics, will be discussed.

Development of a disaggregation-induced emission probe for the detection of RecA inteins from Mycobacterium tuberculosis.

A fluorescent sensor - InR - with disaggregation-induced emission (DIE) characteristics has been developed for the label free detection of RecA inteins from Mycobacterium tuberculosis. Mechanistic studies demonstrate that InR binds at a hydrophobic pocket of RecA inteins, which is constituted by Gly435, Glu434, Arg405 and Arg 7, resulting in the disaggregation and recovery of its fluorescence.

High-throughput screening for RecA inhibitors using a transcreener adenosine 5'-O-diphosphate assay.

The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5'-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener(®) adenosine 5'-O-diphosphate [ADP](2) fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z'=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC(50) values ≤ 10 µM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays.

Methods to classify bacterial pathogens in cystic fibrosis.

Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch's criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients.

Bacillus subtilis CinA is a stationary phase-induced protein that localizes to the nucleoid and plays a minor role in competent cells.

CinA is a conserved bacterial protein that has been reported to play an important role during competence in Streptococcus pneumoniae by recruiting the RecA protein to the cell membrane. Here, we provide information on the homologous CinA in Bacillus subtilis. We found that the synthesis of CinA is upregulated during stationary phase in all cells. The loss of CinA has a mild effect during competence, but it has no influence on the localization of RecA. CinA was observed to be associated with the nucleoid in the cell, and not with the cell membrane, as shown for S. pneumoniae. Purified CinA is a soluble protein, probably forming trimers, like other homologues, which share a domain with CinA that has been reported to be involved in molybdopterine biosynthesis. Our results suggest that CinA plays a nucleoid-associated general role in cells entering stationary phase that is not specific to competence in B. subtilis and possibly in many other bacteria.

Optical tracking of a stress-responsive gene amplifier applied to cell-based biosensing and the study of synthetic architectures.

A synthetic regulatory construct based on a two-stage amplifying promoter cascade is applied to whole-cell biosensing. Green fluorescent protein (GFP) and red fluorescent protein (RFP) enable two-component tracking of the response event, enabling the system to exhibit increased sensitivity, a lower limit of detection, and a unique optical density-free assessment mode. Specifically, the recA and tac promoters are linked by the LacI repressor in Escherichia coli, where DNA-damage activates the recA promoter and the up-regulation of GFP and LacI proteins. LacI represses the tac promoter, down-regulating the otherwise constitutive mCherry transcription. The response of the construct was compared with two singly tagged, conventional recA promoter-reporter constructs: recA::gfpmut3.1 and recA::mCherry. Using a miniature LED-based flow-through optical detector developed for remote sensing applications, limits of detection for the dual reporter construct reached as low as 0.1 nM MMC. By comparison, single-ended reporters recA::mCherry and recA::gfpmut3.1 achieved best limits of detection of 0.25 nM and 2.0 nM, respectively. An approach to three-component optical analysis, based on a system of detectors with coupled calibration equations enables accurate assessments of the red fluorescence, green fluorescence, and biomass of sensor cell suspensions. The system approach is effective at overcoming interferences caused by optically dense samples and overlapping fluorescence spectra. Such a technique may be useful in studying the biological mechanisms which underlie the synthetic regulatory device of this work and others.

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA.

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell's biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be approximately 10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3'- or 5'-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.

A novel SMC-like protein, SbcE (YhaN), is involved in DNA double-strand break repair and competence in Bacillus subtilis.

Bacillus subtilis and most Gram positive bacteria possess four SMC like proteins: SMC, SbcC, RecN and the product of the yhaN gene, termed SbcE. SbcE is most similar to SbcC but contains a unique central domain. We show that SbcE plays a role during transformation in competent cells and in DNA double-strand break (DSB) repair. The phenotypes were strongly exacerbated by the additional deletion of recN or of sbcC, suggesting that all three proteins act upstream of RecA and provide distinct avenues for presynapsis. SbcE accumulated at the cell poles in competent cells, and localized as a discrete focus on the nucleoids in 10% of growing cells. This number moderately increased after treatment with DNA damaging agents and in the absence of RecN or of SbcC. Damage-induced foci of SbcE arose early after induction of DNA damage and rarely colocalized with the replication machinery. Our work shows that SMC-like proteins in B. subtilis play roles at different subcellular sites during DNA repair. SbcC operates at breaks occurring at the replication machinery, whereas RecN and SbcE function mainly, but not exclusively, at DSBs arising elsewhere on the chromosome. In agreement with this idea, we found that RecN-YFP damage-induced assemblies also arise in the absence of ongoing replication.

Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry.

A miniature flow-through optical cell has been developed with the potential for integration into a stand-alone, potentially disposable whole-cell biosensor platform. The compact and inexpensive optical system is comprised of closely coupled light-emitting diodes (LEDs), light-to-frequency (LTF) photodiodes, and celluloid filters. The system has been optimized to measure fluorescent reporters produced by cultures of biosensor cells in liquid suspension. As demonstration subjects, Escherichia coli cells carrying medium-copy plasmids with fluorescent reporter fusions to the rec promoter were exposed to the DNA-damaging agent mitomycin C (MMC). As reporter proteins, green fluorescent protein (GFP) and red fluorescent protein (RFP) were compared for suitability in the compact instrument. The RFP mCherry outperformed GFP (GFPmut3.1) as a reporter protein in the developed system on two counts. First, measurement distortions due to high optical density suspensions are minimal using RFP compared to GFP. Second, the limit of detection for MMC is estimated at 0.25nM for recA::mCherry and 2.0nM for recA::gfpmut3.1. Finally, a measurement method is presented whereby multiple channels of optical data are calibrated in an integrated fashion to allow simultaneous measurement of fluorescence and biomass concentration. The method substantially eliminates optical distortions due to dense samples and thus obviates the conventional need for sample dilution prior to measurement.

Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity.

The diazotrophic bacteria collectively known as "rhizobia" are important for establishing symbiotic N(2)-fixing associations with many legumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptable yields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNA gene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis (MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12 Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK, gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common bean ranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common bean also formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutual agreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broad genetic diversity among strains classified as Rhizobium tropici, providing evidence of new species.

DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells.

BACKGROUND: DprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown. RESULTS: We show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein. CONCLUSION: Our data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA.

A complementary pair of rapid molecular screening assays for RecA activities.

The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index.

Involvement of mitochondrial-targeted RecA in the repair of mitochondrial DNA in the moss, Physcomitrella patens.

Homologous recombination is a universal process that contributes to genetic diversity and genomic integrity. Bacterial-type RecA generally exists in all bacteria and plays a crucial role in homologous recombination. Although RecA homologues also exist in plant mitochondria, there have been few reports about the in vivo functions of these homologues. We identified a recA gene orthologue (named PprecA1) in a cDNA library of the moss, Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA1 to GFP caused a targeting of PpRecA1 to mitochondria. PprecA1 partially complemented the effects of a DNA damaging agent in an Escherichia coli recA deficient strain. Additionally, the expression of PprecA1 was induced by treating the plants with DNA damaging agents. Disruption of PprecA1 by targeted replacement resulted lower rate of the recovery of the mitochondrial DNA from methyl methan sulfonate damage. This is the first report about the characteristics of a null mutant of bacterial-type recA gene in plant. The data suggest that PprecA1 participates in the repair of mitochondrial DNA in P. patens.

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene.

Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum lexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the lexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.