Antisera against flagellin A or B inhibits Pseudomonas aeruginosa motility as measured by novel video microscopy assay.

Flagellum-mediated motility is essential to Pseudomonas aeruginosa (P. aeruginosa) virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin P. aeruginosa motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing P. aeruginosa strain PAO1 (FlaB+) and GFP-expressing P. aeruginosa strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased P. aeruginosa motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.

Dethiosulfovibrio faecalis sp. nov., a novel proteolytic, non-sulphur-reducing bacterium isolated from a marine aquaculture solid waste bioreactor.

A new Dethiosulfovibrio strain, designated F2BT, was isolated from an anaerobic digester for treating solid waste from a marine recirculating aquaculture system. The motile, Gram-negative, non-spore-forming curved rods were 2-7 µm long and 1 µm in diameter. Growth occurred at temperatures ranging from 20 to 40 °C with a maximum rate of growth at 30 °C. The pH range for growth was pH 6.0-8.0, with a maximum rate of growth at pH 7.5. This isolate was halotolerant growing in NaCl concentrations ranging from 0 to 1.6 M with a maximum rate of growth at 0.4 M. Similarly to the five described Dethiosulfovibrio species, this obligate anaerobe isolate was fermentative, capable of utilizing peptides, amino acids and some organic acids for growth, but unlike described strains in the genus did not reduce thiosulphate or elemental sulphur to hydrogen sulphide during fermentation of organic substrates. The G+C content of 55 mol% is similar to the described Dethiosulfovibrio species. The average nucleotide identity analysis between whole genome sequences showed less than 93.15% sequence similarity between strain F2BT and the five other described Dethiosulfovibrio species. Differences in the physiological and phylogenetic characteristics between the new strain and other Dethiosulfovibrio specied indicate that F2BT represents a novel species of this genus and the epithet Dethiosulfovibrio faecalis sp. nov. is proposed. The type strain is F2BT (=DSM 112078T=KCTC25378T).

MrpA functions in energy conversion during acetate-dependent growth of Methanosarcina acetivorans.

The role of the multisubunit sodium/proton antiporter (Mrp) of Methanosarcina acetivorans was investigated with a mutant deleted for the gene encoding the MrpA subunit. Antiporter activity was 5-fold greater in acetate-grown versus methanol-grown wild-type cells, consistent with the previously published relative levels of mrp transcript. The rate, final optical density, and dry weight/methane ratio decreased for the mutant versus wild type when cultured with a growth-limiting concentration of acetate. All growth parameters of the mutant or wild type were identical when grown with methanol in medium containing a growth-limiting Na(+) concentration of 1.04 M. The lag phase, growth rate, and final optical density for growth of the mutant were suboptimal compared to the wild type when cultured with acetate in medium containing either 0.54 or 1.04 M Na(+). The addition of 25 mM NaCl to resting cell suspensions stimulated ATP synthesis driven by a potassium diffusion potential. ATP synthesis was greater in wild-type than mutant cells grown with acetate, a trend that held for methanol-grown cells, albeit less pronounced. Both sodium and proton ionophores reduced ATP synthesis in the wild type grown with either substrate. The results indicated that the Mrp complex is essential for efficient ATP synthesis and optimal growth at the low concentrations of acetate encountered in the environment.

Desiccation as a long-term survival mechanism for the archaeon Methanosarcina barkeri.

Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures.

A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans.

BACKGROUND: Correct annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability. RESULTS: The target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed. CONCLUSIONS: In all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.

Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14.

The modified nucleosides N(2)-methylguanosine and N(2)(2)-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m(2)G at position 6 in tRNA(Cys). The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m(2)G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20-30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m(2)G6 tRNA modification.

A 5' leader sequence regulates expression of methanosarcinal CO dehydrogenase/acetyl coenzyme A synthase.

In vivo expression of CO dehydrogenase/acetyl coenzyme A synthase in Methanosarcina spp. is coordinately regulated in response to substrate by at least two mechanisms: differential transcription initiation and early elongation termination near the 3' end of a 371-bp leader sequence. This is the first report of regulation of transcription elongation in the Archaea.

The archetype gamma-class carbonic anhydrase (Cam) contains iron when synthesized in vivo.

A recombinant protein overproduction system was developed in Methanosarcina acetivorans to facilitate biochemical characterization of oxygen-sensitive metalloenzymes from strictly anaerobic species in the Archaea domain. The system was used to overproduce the archetype of the independently evolved gamma-class carbonic anhydrase. The overproduced enzyme was oxygen sensitive and had full incorporation of iron instead of zinc observed when overproduced in Escherichia coli. This, the first report of in vivo iron incorporation for any carbonic anhydrase, supports the need to reevaluate the role of iron in all classes of carbonic anhydrases derived from anaerobic environments.

Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans.

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.