Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.     

Diagnosis of early pregnancy by ultrasound in Macaca fascicularis

Real-time ultrasonography was used to detect early pregnancy in 32 longtailed macaques (Macaca fascicularis). In 92% of the successful conceptions, a correct diagnosis was made. The earliest sign of pregnancy was an intrauterine ringlike structure (11 days). A "line swelling" (14 days) preceded definite fetal echoes (21 days), and fetal heart motion (30 days) proved fetal viability. Ultrasound is a rapid, noninvasive, and relatively cost-effective method of diagnosing and monitoring early pregnancy in M. fascicularis.     

Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts

Wound healing in Swiss 3T3 cultures was investigated with video-enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wound was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of approximately 0.26 microns/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 microns in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of approximately 0.21 microns/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.     

Tools and methodologies to support more sustainable biofuel feedstock production

Increasingly, government regulations, voluntary standards, and company guidelines require that biofuel production complies with sustainability criteria. For some stakeholders, however, compliance with these criteria may seem complex, costly, or unfeasible. What exiting tools, then, might facilitate compliance with a variety of biofuel-related sustainability criteria? This paper presents four existing tools and methodologies that can help stakeholders assess (and mitigate) potential risks associated with feedstock production, and can thus facilitate compliance with requirements under different requirement systems. These include the Integrated Biodiversity Assessment Tool (IBAT), the ARtificial Intelligence for Ecosystem Services (ARIES) tool, the Responsible Cultivation Areas (RCA) methodology, and the related Biofuels + Forest Carbon (Biofuel + FC) methodology.     

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.     

Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-¹⁴C]glucose and [2-¹⁴C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-¹⁴C]glucose released [¹⁴C]acetate and ¹⁴CO₂ as the main products, with [¹⁴C]acetate being released faster than ¹⁴CO₂. The products of [2-¹⁴C]acetate metabolism, on the other hand, were ¹⁴CO₂ during the reduction phase of soil incubation (days 0 to 15) and ¹⁴CH₄ during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH₄ was produced from acetate, of which up to 56% was produced from glucose degradation.     

Loss of the SPHF homologue Slr1768 leads to a catastrophic failure in the maintenance of thylakoid membranes in Synechocystis sp. PCC 6803

Background

In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified.

Methodology/Principal Findings

We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Δslr1768 mutant with the wild-type, we found that Δslr1768 has a conditional phenotype; specifically under high light conditions (130 µmol m⁻² s⁻¹) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 µmol m⁻² s⁻¹) the phenotype is significantly reduced, with a growth rate similar to the wild-type and only a low frequency of cells with evident thylakoid disruption.

Conclusions/Significance

This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids.     

Site-specific properties of Tn7 transposition into the E. coli Chromosome

Summary A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.     

FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp PCC 6803

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.     

Diterpenoids from Ajuga chamaepitys: Two neo-clerodane derivatives

From the whole plant of Ajuga chamaepitys two new neo-clerodane diterpenoids, ajugapitin and its dihydro derivative, have been isolated. Their