S U M M A R YStarved cells of Candida utilis accumulated Zn2+ by two different processes. The first was a rapid, energy-and temperature-independent system that probably represented binding to the cell surface. The cells also possessed an energy-, pH-, and temperature-dependent system that was capable of accumulating much greater quantities of the cation than the binding process. The energy-dependent system was inhibited by KCN, Na,HAsO,, m-chlorophenyl cartionylcyanide hydrazone, N-ethylmaleimide, EDTA and diethylenetriaminepenta-acetic acid. The system was specific inasmuch as Ca2+, Cr3+, Mn2+, Co2+ or Cu2+ did not compete with, inhibit, or enhance the process. Zn2+ uptake was inhibited by Cd2+. The system exhibited saturation kinetics with a half-saturation value of 1-3 ,UM and a maximum rate of 0.21 (nmol Zn2+) min-l (mg dry wt-l) at 30 "C. Zn2+ uptake required intact membranes since only the binding process was observed in the presence of nystatin, toluene, or sodium dodecyl sulphate. Cells did not exchange recently accumulated 65Zn following the addition of a large excess of non-radioactive Zn2+. Similarly, cells pre-loaded with 65Zn did not lose the cation during starvation, and efflux did not occur when glucose and exogenous Zn2+ were supplied after the starvation period. Efflux was only observed after the addition of toluene or nystatin, or when cells were heated to IOO "C. Cells fed a large quantity of Zn2+ contained a protein fraction resembling animal cell metallothionein. In batch culture, cells of C. utilis accumulated Zn2+ only during the lag phase and the latter half of the exponential-growth phase.

No abstract

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxiceils. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pl, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. paUidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.

A phosphatase specific for the hydrolysis of 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated threeto fourfold by the addition of 1.0 mM Co' or Mg2" and to a lesser extent by 1.0 mM Ba2 , Zn2+, and Mn2. The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu2+, Ca2+, Cd2+, Hg2+, and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 ± 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent Km for KDO-8-phosphate was determined to be 5.8 ± 0.9 x 10' M in the presence of 1.0 mM Co +, 9.1 ± 1 x 10' M in the presence of 1.0 mM Mg2+, and 1.0 ± 0.2 x 10-4 M in the absence of added Co2+ or Mg2+. 3-Deoxy-D-manno-octulosonic acid (KDO) is a unique sugar that is an integral part of the lipopolysaccharide region of most gram-negative bacteria. This eight-carbon keto sugar is the direct link between lipid A and the growing polysaccharide chain (10, 23, 25, 26). Osborn and co-workers (16, 20, 24, 26) have isolated temperature-sensitive mutants defective in KDO biosynthesis and demonstrated the involvement of KDO in the maturation of lipid A. The biosynthesis and utilization of KDO is known to involve at least five sequential reactions: D-ribulose 5-phosphate D-arabinose 5-phosphate D-arabinose 5-phosphate + phosphoenolpyru-(2) vate (PEP)-KDO-8-phosphate + Pi KDO-8-phosphate-* KDO + Pi