The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-terminal repeat domain (CTD) of RNA polymerase II was developed and used to identify one human phosphoCTD-associating protein as CA150. CA150 is a nuclear factor implicated in transcription elongation. Because the hyperphosphorylated CTD is a feature of actively transcribing RNA polymerase II (Pol II), phosphoCTD (PCTD) binding places CA150 in a location appropriate for performing a role in transcription elongation-related events. Several recombinant segments of CA150 bound the PCTD. Predominant binding is mediated by the portion of CA150 containing six FF domains, compact modules of previously unknown function. In fact, small recombinant proteins containing the fifth FF domain bound the PCTD. PCTD binding is the first specific function assigned to an FF domain. As FF domains are found in a variety of nuclear proteins, it is likely that some of these proteins are also PCTD-associating proteins. Thus FF domains appear to be compact protein-interaction modules that, like WW domains, can be evolutionarily shuffled to organize nuclear function. T he C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is an unusual protein moiety that mediates critical interactions between the core transcription machinery and other nuclear factors. The mammalian CTD is a conserved Pol II-specific domain consisting of 52 repeats of the consensus sequence YSPTSPS (1). In its hypophosphorylated form, the CTD interacts with several factors as a central organizer of the ''holoenzyme'' involved in preinitiation complex formation and transcription initiation (2-5). Hyperphosphorylation of the CTD accompanies the transition into active elongation mode (6-9) and heralds dramatic changes in the properties of the transcription complex. Two alterations notable in the current context are an increase in Pol II's elongation competence (e.g., 10, 11-13) and a dramatic change in CTDassociated factors (14).Our current knowledge of factors that associate with the CTD is fragmentary, and in particular proteins that bind the CTD or phosphoCTD (PCTD) directly have been identified in only a few instances. One example is capping enzyme from yeast and human cells, which binds to the phospho-but not the nonPCTD (15-18); incidentally, this discrimination can explain how capping enzymes are targeted specifically to elongating RNA polymerase II. Other examples of demonstrated CTD-binding proteins are the cleavage͞polyadenylation factors CstF and CPSF (19). In the case of CstF, binding appears to be predominantly through the 50-kDa subunit. We recently demonstrated that the unusual peptidyl prolyl isomerase, ESS1͞Pin1, binds tightly to the PCTD via its WW domain (20). This binding could facilitate isomerization of phosphoserine (PSer)-Pro bonds in the CTD by the peptidyl prolyl cis͞trans isomerase domain, altering the conformation of the CTD repeats and modulating the binding of factors to the PCTD.A few additional proteins that bind the CTD directly ...

Palmitoleate is not present in lipid A isolated fromEscherichia coli grown at 30°C or higher, but it comprises ϳ11% of the fatty acyl chains of lipid A in cells grown at 12°C. The appearance of palmitoleate at 12°C is accompanied by a decline in laurate from ϳ18% to ϳ5.5%. We now report that wild-type E. coli shifted from 30°C to 12°C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo 2 -lipid IV A . The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12°C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo 2 -lipid IV A (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12°C.