NATURE. VOL. 220. OCTOBER 19. 1968

Dr J. H. Subak-Sharpe from the Burroughs Wellcome Foundation.

Section on Human Biochemical Genetics, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland.

MRC Experimental Virus Research Unit. University of Glasgow.

Received August 26,1968.

1 Brockman, R. W., Kelley, G . G., Stutts, P., and Copeland, V., Nature,lBl. 469 (1961).

' Szybalski, W., Szybalska, E. H., and Ragni, G. , Nut. Cancer Inst. Monog., 7, 75 (1962).

' Littlefield, J. W., Proc. US Nut. Bead. Sci., 50, 568 (1968). ' Subak-Sharpe, H., Ezp. Cell Res., 38,106 (1965). Veegmiller, J. E., Rosenbloom, F. M., and Kelley, W. N., Science, 155, 1682

(1 967). ~-.. , Rosenbloom, Ic. M., Kelley, W. N., Miller, J., Henderson, J. I?., and

Seegmlller, J. E., J. Amer. Med. Assoc.,202,175 (1967). Subak-Sharpe, H., Biirk, R. R., and Pitts, J. D., Heredity, 21, 342 (1966). Subak-Sharpe, H., Biirk, R. R., and Pitts, J. D., J. Cell Sci. (in the press). Bark, R . R., Pitts, J. D., and Subak-Sharpe, H., Ezp. Cell Res. (in thc

press). Stoker, M. G. P., E m . Cell Rex., 85, 429 (1964).

I ' Hill, M., and Spurn&, V., Em. Cell Res., 50 , 208 (1968). Fujimoto, ,W. Y., Seegmiller, J. E., Uhlendorf, R . W., and Jacobson, C' . B.,

Lancet ( ~ n the press).

Dissolved Organic Carbon from Deep Waters resists Microbial Oxidation OCEANS contain a large absolute amount of organic material in solution. The origin, fate and involvement in biogeochemical processes of this large, but extremely dilute, pool of organic compounds is an enigma to those studying the life and chemistry of the oceans. Recent studies by several workers1-' have shown that the dissolved organic carbon (DOC) content of deep water is relatively constant over a wide range of depths and throughout rnany regions of the world's oceans. The DOC content averages about 0.60 mg/l. and does not decrease signifi- cantly with depth from 400 m to 6,000 m (refs. 2 and 4), nor does the DOC increase in deep waters below highly productive regions such as along the coast of Peru2s4. The range of reported values is from 0.40 to 0-70 mg/l.

In the work ~ i t e d l - ~ the DOC was measured by the wet oxidation method of Menzel and Vaccaros. In this method the organic compounds are oxidized in sealed glass ampoules by persulphate and the resultant CO, is measured by infrared spectrometry. The method of Menzel and Vaccaros agrees well with results obtained by photo-oxidation with ultraviolet irradiation9, contra- indicating that the wet oxidation incompletely oxidizes any of the organic material in seawater. It remains to be settled why S k o p i n ~ t e v ~ ~ - l ~ , using dry combustion, con- sistently measures two to three times more DOC than is measured by wet or photo-oxidation.

The relative constancy of the distribution of DOC over great depths and wide geographic areas suggests that dissolved organic material in deep water is biochemically inert. If there were significant turnover of DOC, one would expect to see a gradient of decreasing abundance away from the surface where new organic matter is syn- thesized. Several ideas have been proposed to explain the apparent biochemical inertness of the organic solutes in deep water: the compounds remaining after exposure to heterotrophic micro-organisms a t the surface could be largely refractory compounds that resist further break-

down1.10~n,13; the concentration of the DOC substrates may be below the threshold concentration necessary for bacterial growthleJ7; or else the lack of particulate surfaces may prevent microbial activity, especially at low concentrations of organic solute^^^.^^.

To test the effect of dilution and surface area on miero- bial degradation of the DOC of deep water, an experiment using pressure dialysis to increase the concentration of DOC and small bottles to provide surfaces for microbial growth was carried out. Water from the depths was collected a t four stations (Table 1) in the western North Atlantic on Cruise 72 of the R/V Chain. The water was collectcd in a 10 1. non-toxic glass and 'Teflon' sampler. Samples for DOC analyses were treated and sealed into ampoules within 1 h of collection. The water to be con- centrated was placed in a 'Diaflo @' ultrafiltration pressure cell over a 'DiafloB UM-3' diffusive membrane. According to the manufacturer, solutes with a higher molecular weight than 500 are retained by this membrane. A pressure of 100 p.s.i. was placed on the system with oxygen. After a two-seven-fold concentration, a sample was taken for an initial concentrated DOC measurement (column 4, Table 1) and the remaining volume removed from above the membrane and placed in combusted glass bottles.

Table 1. CHANGE IN DISSOLVED ORGANIC CAEBON (DOC) AFTER CONCENTRA- TION AND INCUBATION IN THE D.4RK AT 20' C

Conc. Conc. Conc. DOC DOC

Location Depth DOC DOC + l mth. + 2 mths. m mg/1. mgjl. mgjl. mgll.

The organic solutes were coucentrated by pressiue dialysis using a diffusive membrane which retained molecules with a molecular weight greater than 500.

The water was not filtered or otherwise treated in an effort to ensure that the original microbial flora was pre- sent and as little disturbed as possible in the conccntrate. The volume concentrated was approximately 1 l., ensuring that some microbes would be present in the concentrate. The concentrate was saturated with oxygen, and subse- quently incubated a t 20' C in the dark for 1 or 2 months, and had optimal conditions for microbial growth. The concentrate was incubated in 125 ml. bottles so that the micro-organisms had large surface areas to grow on. During incubation the presence of viable microbes was tested with Difco AC medium rehydrated with autoclased seawater. Surface water from Vineyard Sound, nea~r Woods Hole, Massachusetts, was treated identically.

The results of the DOC measurements a t all the stations occupied on Cruise 72 are given in Fig. 1; the water con- tained an avcragc of 0.65 mg of carbon/l. from 300 m to 4,900 m. These results agree well with Menzel'sz earlier n~easurements in this region. Deep water from four stations was concentrated until it contained from 2.7 to 4.1 mg/l. of DOC. This degree of concentration was chosen for two reasons: first, studies showed that indigenous microbes could consume DOC from surface waters when present in these concentrations, and second, the diffusive membrane retention characteristics were somewhat dependent on concentration; that is, 98 per cent of the DOC was retained during a 3.6-fold concentration but only 72 per cent was retained during a 7.4-fold concentra- tion. This characteristic made use of high concentration factors impossible without the undesirable feature of selectively concentrating the larger molecular weight solutes.

Table 1 shows that when DOC from surface water was concentrated to 4.2 mg/l. the microbial flora decreased the organic carbon to 2.1 mg/l. in one month; about

NATURE. V O L 220. OCTOBER 19. 1968

DOC (mg/l.) 0.40 0.80 1.20

Fig. 1. Composite vertieal proflle of the distribution of dissolved organic carbon in the western North Atlantic Ocean on a section from 40' 43' N

and 69" 11' W to 35" 25' Nand 67" 17' W.

80 per cent of the concentrated solute was consumed and the amount of DOC remaining was approximately the same as in the original unconcentrated water.

In contrast to this result there was no change in the amount of DOC in any of the deep r wateconcentrates after 1 or 2 months (Table 1) . The Difco AC medium assay established that viable microbes were present in each concentrate and the microbial activity in the surface concentrates showed that there is no inherent toxicity in the concentration procedure. These experiments can therefore be assumed to test whether low concentration and/or lack of surface area prevents the microbial mineral- ization of deep water organic solutes. The absence of microbial oxidation of deep water DOC after concentration to 2 . P 3 . 7 mg/l. reported here provides experimental evidence for the idea that organic material dissolved in the deep sea is a class of compounds or complexes of compounds which are relatively resistant to biochemical oxidation.

This work was supported by a grant from the US National Science Foundation.

RICHARD T. BARBER Woods Hole Oceanographic Institution, Woods Hole, Massachusetts.

Received August 8, 1968.

Nenzcl, D. W., Deep-Sea Res . , l l , 757 (1964). Menxel, D W., Deep-Sea Res., 14, 228 (1967). ' Menzel, D. W., and Ryther, J. H., Deep-Sea Res., 16, 32 i (1968). ' Barber, R . T., theais, Stanford Univ., 132 (1967). ' \Villiams, P. W., Nature,ZlB, 152 (1968). Wgura, N.. and Hanya, T., J. Oceanol. Limnol..l, 91 (1967). ' Holm-Hans~n, O. , Strickland, J. D. H., and Williams, P. M., Limnol.

Oceanogr ,11, 545 (1966). Menzel, D. W., and Vaccaro, 11. F., Lamnol. Oceanog?., 9, 138 (1964). Armstrong, F. A. J., Williams, P. W., and Strickland, J. D. H., Nature,211,

479 (1966). 'OSkopinstev, B. A., Acad. Sci. USSIl, Trans. Mar. Hydrophys. Znst., 19, 1

(1960). " Skopinstev, B. A, , Oceanology, 6 , 361 (1966).

la Skopinstev, B. A., Abs. Second Intern. Oceanogr. Congr., 340 (1966). la Vaccaro, R. F., and Jannasch, H. W., fimnol. Oceanogr.,ll, 596 (1966). l4 Duursma, E. K., Neth. J . Sea Res., 2.85 (1961). lVaunasch, H. W., J. Cen. Microbial., 18, 609 (1958). lVaunasch, H. W., Limnol. O c e a w . , U, 264 (1967). l7 Jannasch, H. JV., in Organic Matter in Natwal Waters (in the prpns).

Mutant of E. coli containing an Altered D NA-dependent R N A Polymerase IT is generally believed that genetic transcription is carried out by an enzyme, DNA-dependent RNA poly- merase', which is capable of copying DNA sequences into RNA sequences. The enzyme requires DNA and the four naturally occurring ribonucleotide triphosphates to pro- duce RNA which is a faithful copy of the DNA templatea in terms of base composition, nearest. neighbour analysis and DNA-RNA hybridization3.

The i n vitro reaction mimics the i n vivo synthesis of RNA in several ways: in both cases the RNA product is asymmetric4j5, because for given regions of the DNA only one strand is transcribed, arid the direction of growth of the RNA chain is from 5' to 3' (refs. 6 and 7).

The study of a mutant with an altered enzyme may make a contribution to the understanding of transcrip- tions, so we began to search for such a mutant and we now report the isolation of an E. coli strain with an altered RNA polymerase.

Through Professor L. Silvestri we recently became aware of the existence of an antibiotic, rifamycin8, isolated from Streptomyces mediterraneus, and of a large variety of semi-synthetic derivatives. Several investigators have reported on the i n vivo mode of action of rifamycin; in particular, Lancini and Sartori, in a detailed study (unpublished), showed that 1%-uracil incorporation in E. coli a t 37" C is blocked 30 s after the addition of rif- ampicin (a semi-synthetic derivative of rifamycin), 14C-phenylalanine incorporation stops after approximately 8 min, but DNA synthesis, on the contrary, continues to termination of the initiated round of replication. These data and those of others1° provide evidence that the primary i n vivo effect of the drug is the inhibition of RNA synthesis.

Hmtmann et al.ll, Wehrli et a1.12 and Umezawa et al.13 have described the inhibition by rifamycin of the i n witro RNA synthesis catalysed by E. coli RNA polymerase. Hartmann et al.ll have suggested that rifamycin acts on the enzyme rather than on the DNA template, like a.ctinomycin.

We have isolated n derivative of E. coli HfrC met-RCrel resistant to rifampicin. Our mutant grew on trypton plates containing 200 ~ g / m l . of the drug. (The wild type is inhibited a t 2 yglml.) We purified the RNA poly- merase from the mutant and from the wild type according t,o Chamberlin and Berga, and Fig. 1 shows the behaviour of.the two purified enzymes when tested a t different con- centrations of rifamycin B using T4 DNA as a template. The mutant preparation is remarkably more resistant to the drug than the enzyme derived from the wild type, and the same level of resistance can be observed through t,he purification steps used in the adopted procedure.

We conclude therefore that our mutant possesses an altered RNA polymerase. In addition, our results definitely demonstrate that the antibiotic acts on t,he enzyme rather than on the DNA; in agreement with this conclusion, binding of 14C-rifampicin to RNA polymerast: has been detected (Di Mauro, E., Marino, P., and Tocchini- Valentini, G. P., unpublished results).

The location of rifampicin resistance (rifR) on the E. coli genetic map should, on the basis of our results, cor- respond to a gene controlling the structure of the enzyme RNA polymerase. We have crossed our mutant (HfrC