Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C1 compounds

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C(1) units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine-glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine-glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine-glyoxylate aminotransferase. 4. The activity of serine-glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine-glyoxylate aminotransferase is necessary for growth on C(1) compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of alpha-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C(1) compounds.     

Oxygenation of methane by methane-utilizing bacteria.

1. Experimental conditions have been found in which small amounts of methanol (approximately 2.5mm) accumulated when washed cell suspensions of methane-grown Pseudomonas methanica and Methanomonas methanooxidans were incubated with methane+oxygen mixtures in Warburg flasks. 2. The methanol formed could be separated completely from water by fractional distillation through glass helices followed by gas chromatography using 20% polyethylene glycol 400 on a Celite 545 support. 3. By using ¹⁸O-enriched oxygen gas the abundance of ¹⁸O in the methanol formed from oxidation of methane was measured with a Perkin–Elmer 270 combined gas chromatograph/mass spectrometer. The results showed that the oxygen in methanol was derived exclusively from gaseous oxygen in both micro-organisms. 4. Control experiments using [¹⁸O]water in incubation mixtures confirmed that there was negligible incorporation of the oxygen atom from water into methanol.     

Microbial growth on C1 compounds. Synthesis of cell constituents by methane- and methanol-grown Pseudomonas methanica

1. A study has been made of the incorporation of carbon from [¹⁴C]methane, [¹⁴C]methanol and [¹⁴C]bicarbonate by cultures of Pseudomonas methanica growing on methane, and [¹⁴C]methanol by cultures of the same organism growing on methanol. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compound for periods up to 3min., has been analysed by chromatography and radioautography. 3. Over 90% of the radioactivity fixed from [¹⁴C]methane or [¹⁴C]methanol at the earliest times of sampling appeared in phosphorylated compounds. Glucose phosphate and fructose phosphate together constituted the largest part of the radioactive phosphates (70–90%); phosphoglycerate was a relatively minor component (2–17%). Other compounds becoming labelled during the incubation included glycine, serine, glutamate, aspartate, malate, citrate and alanine. 4. The first stable products of [¹⁴C]bicarbonate fixation were malate and aspartate (containing between them over 90% of the total radioactivity fixed at the earliest times of sampling). 5. The percentage of the total radioactivity fixed that was contained in each of the radioactive compounds has been plotted against time. The slopes of the curves obtained show that hexose phosphates are primary stable products of [¹⁴C]methane and [¹⁴C]methanol incorporation and that aspartate and malate are primary stable products of [¹⁴C]bicarbonate incorporation. 6. No carboxydismutase activity has been found in cell-free extracts of the organism. This fact, together with the other findings, shows that an autotrophic metabolism involving the ribulose diphosphate cycle of carbon dioxide fixation cannot be operating.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Synthesis of cell constituents by methane-grown Methylococcus capsulatus and Methanomonas methanooxidans

1. A study was made of the incorporation of carbon from [(14)C]methanol by cultures of Methylococcus capsulatus and Methanomonas methanooxidans growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled substrate for periods of up to 3min, was analysed by chromatography and radioautography. 3. Over 80% of the radioactivity fixed by Methylococcus capsulatus at 30 degrees C at the earliest times of sampling appeared in phosphorylated compounds, of which glucose phosphate constituted 60%. 4. Most of the radioactivity fixed by Methanomonas methanooxidans at 30 degrees C at the earliest times of sampling appeared in serine, malate, aspartate and an unknown compound(s) tentatively suggested to be folate derivative(s). At 16 degrees C, [(14)C]methanol was fixed predominantly into serine and the unknown compound(s). 5. Extracts of Methylococcus capsulatus contain an enzyme system that catalyses the condensation of formaldehyde and ribose 5-phosphate to give a mixture consisting mainly of fructose phosphate and allulose phosphate. No similar activity was detected in extracts of Methanomonas methanooxidans. A convenient method was developed for assay of this enzyme system. 6. The enzyme system catalysing the condensation of formaldehyde with ribose 5-phosphate is particle-bound in both Methylococcus capsulatus and Pseudomonas methanica and is unstable in the absence of Mg(2+). 7. Extracts of Methanomonas methanooxidans contain high activities of d-glycerate-NAD oxidoreductase, whereas extracts of Methylococcus capsulatus and Pseudomonas methanica contain negligible activities of this enzyme. 8. These results indicate that during growth of Methylococcus capsulatus on methane, as with Pseudomonas methanica, cell constituents are made by the ribose phosphate cycle of formaldehyde fixation. This contrasts with Methanomonas methanooxidans, whose assimilation pathway resembles in some features that of Pseudomonas AM1 growing on methanol.     

Microbial growth on C1 compounds. Incorporation of C1 units into allulose phosphate by extracts of Pseudomonas methanica

1. Incubation of cell-free extracts of methane- or methanol-grown Pseudomonas methanica with [(14)C]formaldehyde and d-ribose 5-phosphate leads to incorporation of radioactivity into a non-volatile product, which has the chromatographic properties of a phosphorylated compound. 2. Treatment of this reaction product with a phosphatase, followed by chromatography, shows the presence of two compounds whose chromatographic properties are consistent with their being free sugars. 3. The minor component of the dephosphorylated products has been identified as fructose. The major component has been identified as allulose (psicose) on the basis of co-chromatography, co-crystallization of the derived phenylosazone and dinitrophenylosazone with authentic derivatives of allulose and behaviour towards oxidation with bromine water. 4. It is suggested that the bacterial extracts catalyse the condensation of a C(1) unit identical with, or derived from, formaldehyde with ribose 5-phosphate to give allulose 6-phosphate. 5. Testing of hexose phosphates and pentose phosphates as substrates has so far shown the reaction to be specific for ribose 5-phosphate. 6. The condensation reaction is not catalysed by extracts of methanol-grown Pseudomonas AM1. 7. A variant of the pentose phosphate cycle, involving this condensation reaction, is suggested as an explanation for the net synthesis of C(3) compounds from C(1) units by P. methanica.     

Technical note: The use of 3D printing in dental anthropology collections

Objectives

Rapid prototyping (RP) technology is becoming more affordable, faster, and is now capable of building models with a high resolution and accuracy. Due to technological limitations, 3D printing in biological anthropology has been mostly limited to museum displays and forensic reconstructions. In this study, we compared the accuracy of different 3D printers to establish whether RP can be used effectively to reproduce anthropological dental collections, potentially replacing access to oftentimes fragile and irreplaceable original material.

Methods

We digitized specimens from the Yuendumu collection of Australian Aboriginal dental casts using a high‐resolution white‐light scanning system and reproduced them using four different 3D printing technologies: stereolithography (SLA); fused deposition modeling (FDM); binder‐jetting; and material‐jetting. We compared the deviations between the original 3D surface models with 3D print scans using color maps generated from a 3D metric deviation analysis.

Results

The 3D printed models reproduced both the detail and discrete morphology of the scanned dental casts. The results of the metric deviation analysis demonstrate that all 3D print models were accurate, with only a few small areas of high deviations. The material‐jetting and SLA printers were found to perform better than the other two printing machines.

Conclusions

The quality of current commercial 3D printers has reached a good level of accuracy and detail reproduction. However, the costs and printing times limit its application to produce large sample numbers for use in most anthropological studies. Nonetheless, RP offers a viable option to preserve numerically constraint fragile skeletal and dental material in paleoanthropological collections.

     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.