Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability.

Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of PtdIns-4,5-bisphosphate (PtdIns[4,5]P2). Hydrolysis of PtdIns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that modulate cell proliferation and differentiation. The PIK1 gene encoding a PtdIns 4-kinase from the yeast Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast (Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Read-through transcription from a derepressed Tn3 promoter affects ColE1 functions on a ColE1::Tn3 composite plasmid.

Mutations in the repressor encoded by the transposon Tn3 tnpR gene lead to increased levels of expression of two gene products: the mutant repressor (TnpR-) and the Tn3 encoded transposase, TnpA (Heffron et al. 1978; Chou et al. 1979a). Derivatives of the ColE1::Tn3 composite plasmid, RSF2124, with mutant Tn3 repressor exhibited the expected elevated levels of transposition. Unexpectedly, hosts containing these tnpR- derivatives produced enhanced levels of the ColE1 encoded toxin, colicin E1. The gene for colicin E1 maps far (0.23-0.98 MU) from the Tn3 insertion point (0.73 MU) (Fig. 1). The colicin E1 overproduction phenotype, designated Eop-, was complemented in trans by wild type repressor gene product (TnpR+) to the wild type phenotype, Eop+. Hosts with RSF2124 derivatives which expressed high levels of both mutant repressor and mutant transposase (TnpR-, TnpA-) were Eop-. Hosts containing plasmids deleted for both tnpA and tnpR promoters were Eop+, while hosts with plasmids carrying a lac promoter substitution for the tnpA promoter were Eop-. These data support the idea that a cis-acting effect of increased transcription from the tnpA promoter into adjacent ColE1 DNA was the cause of colicin overproduction. Increased transcription activated a putative colicin augmentation function (caf) whose presence was required for the Eop- phenotype. Deletion mapping established that one boundary of the caf locus lies within 52 bases of the junction of the left end of Tn3 and ColE1 DNA. ColE1 DNA in this area contains an open reading frame which could encode either a 74 or a 63 residue protein (B. Polisky, unpublished DNA sequence data). The presence of increased levels of an mRNA transcript from this region and/or the increased expression of protein(s) from this transcript could result in an Eop- phenotype. Expression of the Eop- phenotype requires the presence of the host recE gene. Evidence is presented which suggests that the recA repressor, lexA protein, controls expression of the recE gene product, ExoVIII.