Structural basis of the interaction between cyclodipeptide synthases and aminoacylated tRNA substrates.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic ß2 and ß7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.

Hepato and nephroprotective activity of methanol extract of Hygrophila spinosa and its antibacterial potential against multidrug resistant Pandoraea sputorum.

This research was aimed to evaluate the phytochemical profile, bactericidal activity of Hygrophila spinosa against multidrug resistant Pandoraea sputorum and assess their antioxidant competence against various radicals and studied their hepatoprotective and nephroprotective activity on HepG2 and HEK 293 cell line. The results showed that the methanol extract has various phytochemical components with reasonable quantity. Fortunately, the multidrug-resistant P. sputorum was sensitive (22.8 ± 0.2 mm of the zone of inhibition) at 15 mg mL-1 concentration of methanol extract. The higher concentration of phenolic and other phytochemical components, showed significant antioxidant activity against ferric, DPPH, hydroxyl, and ABTS radicals, with IC50 values of 71.09, 64.333, 91.157, and 104.931 g mL-1, respectively. Surprisingly, the methanol extract possesses hepato and nephroprotective activity against CCl4 and cisplatin-induced cytotoxicity on HepG2 and HEK 293 cell lines, respectively. It maintains the cell viability as up to 90.48% and 90.35% of HepG2 and EK 293 cell line at the concentration of 20 µg mL-1. The FTIR analysis states that the methanol extract possesses a significant functional group responsible for these multi-potential activities. These results suggest that, the methanol extract of H. spinosa might contain the most significant bioactive components with outstanding medicinal properties.

Degradation of dibutyl phthalate (DBP) by a bacterial consortium and characterization of two novel esterases capable of hydrolyzing PAEs sequentially.

Phthalate esters (PAEs), a class of toxic anthropogenic compounds, have been predominantly used as additives or plasticizers, and great concern and interests have been raised regarding its environmental behavior and degradation mechanism. In the present study, a bacterial consortium consisting of Microbacterium sp. PAE-1 and Pandoraea sp. PAE-2 was isolated by the enrichment method, which could degrade dibutyl phthalate (DBP) completely by biochemical cooperation. DBP was converted to phthalic acid (PA) via monobutyl phthalate (MBP) by two sequential hydrolysis steps in strain PAE-1, and then PA was further degraded by strain PAE-2. Strain PAE-1 could hydrolyze many dialkyl Phthalate esters (PAEs) including dimethyl, diethyl, dibutyl, dipentyl, benzyl butyl, dihexyl, di-(2-ethyhexyl) and their corresponding monoalkyl PAEs. Two esterase genes named dpeH and mpeH, located in the same transcription unit, were cloned from strain PAE-1 by the shotgun method and heterologously expressed in Escherichia. coli (DE3). The Km and kcat values of DpeH for DBP were 9.60 ± 0.97 µM and (2.72 ± 0.06) × 106 s-1, while those of MpeH for MBP were 18.61 ± 2.00 µM and (5.83 ± 1.00) × 105 s-1, respectively. DpeH could only hydrolyze dialkyl PAEs to the corresponding monoalkyl PAEs, which were then hydrolyzed to PA by MpeH. DpeH shares the highest similarity (53%) with an alpha/beta hydrolase from Microbacterium sp. MED-G48 and MpeH shows only 25% identity with a secreted lipase from Trichophyton benhamiae CBS 112371, indicating that DpeH and MpeH are two novel hydrolases against PAEs.

Epidemic spread of Pandoraea pulmonicola in a cystic fibrosis center.

BACKGROUND: Pandoraea spp. are recently discovered bacteria, mainly recovered from cystic fibrosis (CF) patients, but their epidemiology and clinical significance are not well known. We describe an epidemic spread of Pandoraea pulmonicola from 2009 in our CF center, involving 6 out of 243 CF patients. METHODS: Bacterial identification used amplified ribosomal DNA restriction analysis (ARDRA), MALDI-TOF mass spectrometry (MALDI-TOF MS) and 16S rDNA gene sequencing. The clonal link between strains was assessed with pulsed field gel electrophoresis (PFGE) using XbaI. Clinical data were gathered for all patients. RESULTS: The index case was chronically colonized since 2000. The main hypothesis for this bacterial spread was a droplet cross-transmission, due to preventive measures not being strictly followed. Antibiotic susceptibility testing revealed resistance to beta-lactams, ciprofloxacin and colistin. However, there was susceptibility to trimethoprim-sulfamethoxazole. All patients were chronically colonized with Pseudomonas aeruginosa, and the acquisition of P. pulmonicola resulted in chronic colonization in all patients. Three patients died, and two patients remained clinically stable, whereas one patient had a decline in lung function. CONCLUSIONS: This study, which is the first to describe an epidemic spread of P. pulmonicola, notes the potential transmissibility of this bacterial species and the need for infection control measures.

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

AIMS: Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. METHODS AND RESULTS: The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. CONCLUSIONS: The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. SIGNIFICANCE AND IMPACT OF THE STUDY: D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started.

Aposymbiosis of a Burkholderiaceae-Related Endobacterium Impacts on Sexual Reproduction of Its Fungal Host.

Bacterial endosymbionts inhabit diverse fungal lineages. Although the number of studies on bacteria is increasing, the mechanisms by which bacteria affect their fungal hosts remain unclear. We herein examined the homothallic isolate, Mortierella sugadairana YTM39, harboring a Burkholderiaceae-related endobacterium, which did not produce sexual spores. We successfully eliminated the bacterium from fungal isolates using ciprofloxacin treatment and asexual spore isolation for germinated asexual spores. Sexual spore formation by the fungus was restored by eliminating the bacterium from isolates. These results indicate that sexual reproduction by the fungus was inhibited by the bacterium. This is the first study on the sexual spore infertility of fungal hosts by endofungal bacteria.

Antibiotic resistome profile based on metagenomics in raw surface drinking water source and the influence of environmental factor: A case study in Huaihe River Basin, China.

The contamination with antibiotic resistance genes (ARGs) in raw drinking water source may pose a direct threat to human health. In this study, metagenomics sequencing and analysis were applied to investigate the ARG pattern in 12 drinking water sources in upper and middle reach of Huaihe River Basin, China. Based on the redundant analysis and multi-linear regression model, location, specific microbial taxa, number of livestock and health facilities significantly influenced the ARG profile in drinking water sources. Besides the cluster effect of ARG in samples from plain and bedrock mountain areas, the samples from fracture aquifer areas also showed a distinctive biogeographic pattern with that from porous aquifer areas. Putative ARGs host Opitutus and Flavobacterium were the enriched biomarkers in plain and fracture aquifer area respectively, which mainly carried bacitracin, multidrug, beta-lactam and tetracycline ARGs. This result illuminated that both natural background and anthropogenic activities in the watershed influenced the ARG profile in natural freshwater system significantly. The low MGEs abundance and absence of pathogen revealed a low ARG dissemination risk in sampled drinking water sources, while Polynucleobacter was an abundant ARGs host and was significantly related to the ARG profile, which indicated that specific bacteria was responsible for ARGs propagation and accumulation in surface freshwater system. Further researches are needed to assess human exposure to raw drinking water source and the potential risk, as well as the species interaction in microbial community and its impact on ARG propagation under oligotrophic condition.

Reduction in arsenic toxicity and uptake in rice (Oryza sativa L.) by As-resistant purple nonsulfur bacteria.

This study aimed to investigate the potential of Rhodopseudomonas palustris C1 and Rubrivivax benzoatilyticus C31 to ameliorate As toxicity and to reduce As uptake in rice. Strain C1 was superior to strain C31 for siderophore production. The mixed culture (1: 1) was most effective in reducing the toxicity of As species [As(III) and/or As(V), each 30 mg/l] by yielding maximal germination index that related to α- and ß-amylase activities in two Thai rice cultivars (HomNil: HN and PathumThani 1: PT). Arsenic toxicity to the seed germination followed the order: mixed As species > As(III) > As(V); and the toxicity was reduced in inoculated sets, particularly with a mixed culture. The mixed culture significantly enhanced rice growth under As stress in both rice cultivars as indicated by an increase in the production of chlorophyll a and b, and also supporting the non-enzymatic (carotenoids, lipid oxidation, and nitric oxide) and enzymatic (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase) activities. These were concomitant with productions of 5-aminolevulinic acid, indole-3-acetic acid, exopolymeric substances, and siderophores which significantly reduced As accumulation in treated rice. It can be concluded that the mixed culture has great potential to ameliorate rice from As toxicity by preventing As species entry into rice for enhancing rice growth and also for reducing As accumulation to produce safe rice from rice grown in contaminated paddy fields.

A novel inducible prophage from the mycosphere inhabitant Paraburkholderia terrae BS437.

Bacteriophages constitute key gene transfer agents in many bacteria. Specifically, they may confer gene mobility to Paraburkholderia spp. that dwells in soil and the mycosphere. In this study, we first screened mycosphere and bulk soils for phages able to produce plaques, however found these to be below detection. Then, prophage identification methods were applied to the genome sequences of the mycosphere-derived Paraburkholderia terrae strains BS001, BS007, BS110 and BS437, next to P. phytofirmans strains BS455, BIFAS53, J1U5 and PsJN. These analyses revealed all bacterial genomes to contain considerable amounts [up to 13.3%] of prophage-like sequences. One sequence predicted to encode a complete phage was found in the genome of P. terrae BS437. Using the inducing agent mitomycin C, we produced high-titered phage suspensions. These indeed encompassed the progeny of the identified prophage (denoted ɸ437), as evidenced using phage major capsid gene molecular detection. We obtained the full sequence of phage ɸ437, which, remarkably, had undergone a reshuffling of two large gene blocks. One predicted moron gene was found, and it is currently analyzed to understand the extent of its ecological significance for the host.

Persistent Infection Because of Pandoraea sputorum in a Young Cystic Fibrosis Patient Resistant to Antimicrobial Treatment.

We report the case of a 13-year-old boy with cystic fibrosis with a pulmonary exacerbation concomitant to the first isolation of Pandoraea sputorum. The imipenem and trimethoprim-sulfamethoxazole treatments failed, with persistence of the bacteria, bronchial congestion and a decline in lung function. Pandoraea sp. is rarely isolated, with only 10 cases reported in France in 2011.

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15.

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.

Paralogs of genes encoding metal resistance proteins in Cupriavidus metallidurans strain CH34.

Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34is a well-studied example of a metal-resistant proteobacterium. Genome sequence analysis revealed the presence of a variety of paralogs of proteins that were previously shown to be involved in heavy metal resistance. Which advantage has C. metallidurans in maintaining all these paralogs during evolution? Paralogs investigated belong to the families RND (resistance nodulation cell division) or CHR (chromate resistance). The respective genes were localized by PCR either on one of the two native megaplasmids pMOL28 and pMOL30 of strain CH34, or on its chromosomal DNA. Gene expression was studied by real-time reverse transcriptase PCR and by reporter gene constructs. Genes found to be inducible were disrupted and their contribution to metal resistance measured. When two or three highly related genes were present, usually one was inducible by heavy metals while the other one or two were silent or constitutively expressed. This suggests that C. metallidurans CH34 carries a variety of no longer or not yet used genes that might serve as surplus material for further developments, an advantage that may compensate for the costs of maintaining these genes during evolution.

Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34.

The four replicons of Cupriavidus metallidurans CH34 (the genome sequence was provided by the US Department of Energy-University of California Joint Genome Institute) contain two gene clusters putatively encoding periplasmic resistance to copper, with an arrangement of genes resembling that of the copSRABCD locus on the 2.1 Mb megaplasmid (MPL) of Ralstonia solanacearum, a closely related plant pathogen. One of the copSRABCD clusters was located on the 2.6 Mb MPL, while the second was found on the pMOL30 (234 kb) plasmid as part of a larger group of genes involved in copper resistance, spanning 17 857 bp in total. In this region, 19 ORFs (copVTMKNSRABCDIJGFLQHE) were identified based on the sequencing of a fragment cloned in an IncW vector, on the preliminary annotation by the Joint Genome Institute, and by using transcriptomic and proteomic data. When introduced into plasmid-cured derivatives of C. metallidurans CH34, the cop locus was able to restore the wild-type MIC, albeit with a biphasic survival curve, with respect to applied Cu(II) concentration. Quantitative-PCR data showed that the 19 ORFs were induced from 2- to 1159-fold when cells were challenged with elevated Cu(II) concentrations. Microarray data showed that the genes that were most induced after a Cu(II) challenge of 0.1 mM belonged to the pMOL30 cop cluster. Megaplasmidic cop genes were also induced, but at a much lower level, with the exception of the highly expressed MPL copD. Proteomic data allowed direct observation on two-dimensional gel electrophoresis, and via mass spectrometry, of pMOL30 CopK, CopR, CopS, CopA, CopB and CopC proteins. Individual cop gene expression depended on both the Cu(II) concentration and the exposure time, suggesting a sequential scheme in the resistance process, involving genes such as copK and copT in an initial phase, while other genes, such as copH, seem to be involved in a late response phase. A concentration of 0.4 mM Cu(II) was the highest to induce maximal expression of most cop genes.