Christian Marchal

Pilin expression in Neisseria gonorrhoeae is under both positive and negative transcriptional control.

We have identified two closely linked genes, pilA and pilB, which act in trans on the pilin promoter. pilA‐pilB map downstream of expression loci pilE1 and opaE1 in the gonococcal chromosome. Subcloning data indicate that pilB acts negatively on the pilin promoter, and insertional inactivation of pilB results in hyperpiliated gonococci. A pilA clone activates the pilin promoter in Escherichia coli, and a pilA‐/pilA+ heterodiploid gonococcus exhibits a P‐ phenotype. Our inability to obtain simple pilA‐ mutants strongly suggests that pilA is an essential gene in the gonococcus. In an in vitro coupled transcription/translation system, inserts spanning the pilA and pilB region direct the synthesis of two proteins of 40 and 58 kd. DNA sequence analysis shows that the pilA and pilB loci encode proteins of 38.6 kd (with a putative DNA binding domain) and 57.9 kd respectively. The pilA and pilB genes are in opposite orientation relative to each other, and the 5′ ends of the two genes overlap. We discuss how these two loci may interact to control pilin expression in the gonococcus.

Control of pilus expression in Neisseria gonorrhoeae as an original system in the family of two-component regulators

We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression in Neisseria gonorrhoeae. Here we show that PilA and PilB have amino acid sequence similarities with members of the two component sensor‐regulator’family of proteins. PilB has homology with histidine kinase sensors. Alkaline phosphatase fusions to the predicted sensor and transmitter domains are described. Their PhoA activity and cellular location suggest that PilB is inserted in the cytoplasmic membrane and predict periplasmic and cytoplasmic locations for the sensor and the transmitter domains, respectively. PilA has homology with response regulators in its N‐terminal part, and with components of the eukaryotic protein secretory apparatus (SRP 54 and SRP receptor) as well as two Escherichia coli gene products in its C‐terminal part. In particular, it contains a putative GTP‐binding site. Mini‐transposon insertions into different regions of pilA were obtained. The phenotypes and genotypes of these mutants and preliminary biochemical studies of the gene products of two of these mutants lend further support to the hypothesis that PitA is a DNA‐binding response regulator and confirm that it participates in an essential function in the bacterium.

PuIO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli

The PulO protein required for extracellular secretion of pullulanase by Klebsiella oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilin peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Neisseria gonorrhoeae in Escherichia coli. The P. aeruginosa prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE–PhoA hybrids tested. Southern hybridization experiments suggest that a PulO homologue is present in the N. gonorrhoeae chromosome.

Genetic study of a membrane protein: DNA sequence alterations due to 17 lamB point mutations affecting adsorption of phage lambda.

Gene lamB encodes the outer membrane receptor for phage lambda in Escherichia coli K12. We have determined the DNA sequence alterations of 17 lamB point mutations which result in resistance to phage lambda h+. The mutations correspond to four phenotypic classes according to the pattern of growth of three phages which use the lambda receptor: lambda h (a one‐step host‐range derivative of lambda h+), lambda hh* (a two‐step host‐range derivative of lambda h+) and K10 (another lambdoid phage). Fourteen mutations are of the missense type and correspond to Gly to Asp changes distributed as follows. One class I mutation is at position 382 of the mature lambda receptor. Seven class I* mutations, four of which at least are independent, are at position 401. Six independent class II mutations are at position 151. The three other (class III) mutations are of the nonsense type. They change codons TGG (Trp) into TAG (amber) at positions 120 (two mutations) and 351 (one mutation). Implications of these results for the topological organization of the lambda receptor as well as possible reasons for the limited number of altered sites detected are discussed.

Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes

The control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control

SummaryBacteriophage lambda vectors, derived from λplac5 were constructed. Their genomes have only one EcoRI restriction site, located near the end of the β-galactosidase gene. Recombinants, constructed in vitro, having a DNA fragment inserted in the EcoRI site, are lac- and can be easily recognized. Expression of such foreign genes is then under the control of the lac promoter. Mutations Qam73 and Sam7 greatly increase the amount of β-galactosidase synthesized by the vector bacteriophage. The λZEQS vector has been certified B2 (EK2) by the French control commission “Recombinaisons génétiques in vitro”.