Subsea permafrost organic carbon stocks are large and of dominantly low reactivity.

Subsea permafrost carbon pools below the Arctic shelf seas are a major unknown in the global carbon cycle. We combine a numerical model of sedimentation and permafrost evolution with simplified carbon turnover to estimate accumulation and microbial decomposition of organic matter on the pan-Arctic shelf over the past four glacial cycles. We find that Arctic shelf permafrost is a globally important long-term carbon sink storing 2822 (1518-4982) Pg OC, double the amount stored in lowland permafrost. Although currently thawing, prior microbial decomposition and organic matter aging limit decomposition rates to less than 48 Tg OC/yr (25-85) constraining emissions due to thaw and suggesting that the large permafrost shelf carbon pool is largely insensitive to thaw. We identify an urgent need to reduce uncertainty in rates of microbial decomposition of organic matter in cold and saline subaquatic environments. Large emissions of methane more likely derive from older and deeper sources than from organic matter in thawing permafrost.

Characterization of Mycobacterium tuberculosis complex isolates from Greek patients with sarcoidosis by Spoligotyping.

Spoligotyping was undertaken with 38 Mycobacterium tuberculosis isolates from Greek sarcoidosis patients and 31 isolates from patients with tuberculosis. Fifty percent of the isolates from sarcoidosis patients and 16.13% of the isolates from patients with tuberculosis were represented by a unique pattern, whereas the remaining isolates belonged to seven shared types. Interestingly, half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis, most of which pertained to shared spoligotypes.

Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli.

The nucleotide sequences of the glpQ and ugpQ genes of Escherichia coli, which both encode glycerophosphoryl diester phosphodiesterases, were determined. The glpQ gene encodes a periplasmic enzyme of 333 amino acids, produced initially with a 25 residue long signal sequence, while ugpQ codes for a cytoplasmic protein of 247 amino acids. Despite differences in size and cellular location, significant similarity in the primary structures of the two enzymes was found suggesting a common evolutionary origin. The 3' end of the ugpQ gene overlaps an open reading frame that is transcribed in the opposite direction. This open reading frame encodes a polypeptide with an unusual composition, i.e., 46 of the 146 amino acids are Gln or Asn. This polypeptide and the UgpQ protein were identified in an in vitro transcription/translation system as proteins with apparent molecular weights of 19.5 and 27 kDa, respectively.

Membrane biogenesis in Escherichia coli: effects of a secA mutation.

In Escherichia coli K-12, temperature-sensitive mutations in the secA gene have been shown to interfere with protein export. Here we show that the effect of a secA mutation is strongly pleiotropic on membrane biogenesis. Freeze-fracture experiments as well as cryosections of the cells revealed the appearance of intracytoplasmic membranes upon induction of the SecA phenotype. The permeability barrier of the outer membrane to detergents was lost. Two alterations in the outer membrane may be responsible for this effect, namely the reduced amounts of outer membrane proteins, or the reduction of the length of the core oligosaccharide of the lipopolysaccharide, which was observed in phage-sensitivity experiments and by SDS-polyacrylamide gel electrophoresis. Phospholipid analysis of the secA mutant, grown under restrictive conditions, revealed a lower content of the negatively charged phospholipid cardiolipin and of 18:1 fatty acid compared to those of the parental strain grown under identical conditions. These results are in line with the hypothesis that protein export and lipid metabolism are coupled.

Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system.

The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system.

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein.

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.

Nucleotide sequence of the exclusion-determining locus of IncI plasmid R144.

The exclusion-determining locus (exc) of IncI plasmid R144 has been proposed to contain two overlapping genes. The nucleotide sequence of this locus, as presently determined, reveals an open reading frame with a coding capacity of 220 amino acids (aa) and with a promoter located upstream of the translation-initiation region. Consistent with the proposed overlapping gene arrangement, a second putative promoter was found within this coding region. Thus, a polypeptide identical to the 147 C-terminal aa of the larger polypeptide can be expressed from this second promoter. The 3'-noncoding region contains a sequence that is representative for a Rho-independent transcription terminator.

Role of the Arg158 residue of the outer membrane PhoE pore protein of Escherichia coli K 12 in bacteriophage TC45 recognition and in channel characteristics.

In order to study the structure-function relationship of the PhoE protein pore we have isolated five independent, TC45-resistant, phoE mutants all of which appeared to produce normal amounts of an electrophoretically altered PhoE protein, designated as PhoE* protein. Nucleotide sequence analysis of the DNA fragments carrying the mutations showed that the mutations all correspond to a G.C to A.T transition at the same place within the phoE gene resulting in a deduced change of amino acid residue arginine 158 into histidine. This result shows that the arginine 158 residue plays an important role in the interaction of the PhoE protein pore with phage TC45. Moreover, studies on the channel properties of the PhoE* protein showed that the PhoE channel has lost part of its preference for negatively charged solutes, as a result of the arginine to histidine change. The results are discussed in terms of the structure-function relationship of PhoE protein as well as in terms of the topological organization of the protein channel in the outer membrane.

Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm.

A phoE-lacZ hybrid gene encoding the N-terminal 300 amino acid residues of pre-PhoE protein, fused to an almost complete beta-galactosidase molecule was constructed in vitro. Cell fractionation experiments suggested that the hybrid gene product is transported to the outer membrane. However, by using immuno-cytochemical labelling on ultra-thin cryosections it was shown that the hybrid protein accumulated in the cytoplasm. Thus, it appears that: (i) data on the localization of hybrid proteins merely based on cell fractionation experiments are not reliable, and (ii) either the C-terminal 15% of PhoE protein contain information which is essential for transport, or PhoE-LacZ hybrid proteins can never be transported out of the cytoplasm. The implications of these results for current models on the translocation of outer membrane proteins are discussed.

Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae.

In Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The corresponding gene of Enterobacter cloacae was transferred to E. coli K-12 by using RP4::mini Mu plasmid pULB113 and selecting for R-prime plasmids that carry the genes proA and proB, which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by gamma delta insertions. The E. cloacae phoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coli phoE gene. Functionally, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.

Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12.

Like the synthesis of alkaline phosphatase, the synthesis of outer membrane PhoE protein is shown to be dependent on the phoM gene product in phoR mutants of E. coli K12. This phoM gene has been cloned into the multi-copy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The gene was localized on the hybrid plasmids by analysis of deletion plasmids constructed in vitro and of mutant plasmids generated by gamma delta insertions. Interestingly, two of the selected hybrid plasmids contained the entire phoA-phoB-phoR region of the chromosome, as a multiple copy state of these genes results in the constitutive synthesis of alkaline phosphatase. The presence of multiple copies of the phoM gene hardly influences the level of expression of alkaline phosphatase and PhoE protein in a pho+ strain, but significantly increases the levels of these proteins in a phoR mutant strain.

Cloning of phoE, the structural gene for the Escherichia coli phosphate limitation-inducible outer membrane pore protein.

The hybrid plasmid pLC44-11 from the Clarke and Carbon collection, which was known to carry the proA gene, was shown also to contain the phoE gene. In vitro recombination techniques were used to subclone a 4.9-kilobase-pair DNA fragment of pLC44-11 into the plasmid vectors pACYC184 and pBR322. Expression of this fragment in a minicell system showed that it codes for the PhoE protein and for polypeptides with apparent molecular weights of 47,000 and 17,000. These results supply definite proof for the earlier supposition that the phoE gene is the structural gene for the outer membrane PhoE protein. Overproduction of the PhoE protein in a phoS strain resulted in reduced amounts of OmpF and LamB proteins.