- Volume 141, Issue 4, 1995

Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L-fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intac modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed ac}cumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism.

The regeneration of the paracrystalline proteinaceous cell wall (S layer) of the methanogen Methanococcus voltae was examined by electron microscopy. The S layer was removed from the cell surface by exposing the cells to a protoplasting buffer but was completely regenerated by 60-80 min resuspension in a regeneration medium by some repair mechanism (normal cell generation time is 16 h). The protoplast surface appeared in freeze-fracture to be initially smooth and featureless and remained so for the first 20 min in regeneration medium. Thereafter, nascent S layer appeared on the surface in the form of paracrystalline patches of various sizes that were distributed over the entire surface. The patches were closely associated with disturbances of the cell surface curvature which may have been caused by electrostatic interactions. The steps leading to the complete regeneration of the S layer could not be followed due to the sampling times used, but presumably the patches grew large enough to contact neighbouring patches and somehow fused into a coherent layer. Other ultrastructural changes occurred to the cell envelope during S layer regeneration, most notably the recurrence of the intramembrane particles of the concave fracture face of the plasma membrane. Flagella remained attached to the protoplasts and were apparently still functional. The major changes to the protein profile of untreated cells, protoplasts and regenerating protoplasts included the loss and resynthesis of polypeptides at the 76 kDa (S layer polypeptide) and 60 kDa (unknown polypeptide) positions. SDS-PAGE evidence indicated that new S layer polypeptides were synthesized 20 min prior to their appearance as patches on the cell surface. The S layer polypeptide may have been shed from the surface during the early stages of regeneration to form the extracellular sheet-like material observed in large quantity in the regeneration medium. The response of the cell to changes that occur at the cell surface indicates that some feedback mechanism gears the synthesis of S layer and associated envelope components.

During growth on the C1 substrates methanol or methylamine, Paracoccus denitrificans is able to activate the expression of the genes encoding methanol dehydrogenase. In a previous paper the isolation of an operon containing two regulatory genes, mxaYX (formerly known as moxYX) and a third gene mxaZ (formerly known as moxZ) was described. MxaY and MxaX were shown to have homology with the signal sensors and the response regulators, respectively. Here we describe the isolation and characterization of mutants with marked and unmarked mutations in mxaZ, mxaY and mxaX. Expression of the structural mox genes was analysed by measuring the expression of a mxaF (moxF)-lacZ transcriptional fusion in the presence of mxaZ, mxaY, mxaX or combinations of these genes. Mutants that were unable to express mxaX were impaired for growth on methanol, did not synthesize MDH and could not express a mxaF-lacZ transcriptional fusion. This indicates that the response regulator MxaX is essential for expression of the structural mox genes. Mutants that had a deletion in mxaY or both mxaY and mxaZ were able to grow on methanol and were able to regulate the expression of the mxaF-lacZ fusion just like the wild-type. These findings indicate that mxaY + and mxaZ + are not essential for normal C1 regulation. In addition the results suggest that, at least in the absence of the signal sensor MxaY, MxaX can be activated via a different, but parallel, signal transduction pathway.

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacl repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding β-glucuronidase were initially placed under the control of the trc promoter and lacl repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trcllacl system in Synechococcus was determined using β-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISRI1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

The genetic organization of two different 5-nitroimidazole (5-Ni) resistance genes was investigated: nimC and nimD from Bacteroides plasmids plP419 and plP421, respectively. The nimC gene (492 bp) and the nimD gene (495 bp) directed the synthesis of polypeptides with deduced molecular masses of 18·37 kDa and 18·48 kDa, respectively. The predicted proteins showed 67-83% identity and 78-91% similarity with the products of two other nimA and nimB genes previously described and could be derived from a common ancestral gene. An insertion sequence element (IS1170) was identified upstream of the nimC gene. IS1170 is 1604 bp in length and is flanked by imperfect inverted repeats (15 bp). IS 1170 is similar to the Bacteroides insertion sequence element IS942 with an identity of 70% at the nucleotide level. The single copy of IS 1170 present on plasmid plP419 is integrated 24 bp upstream of the initiation codon of nimC. Similar genetic organization was found on plasmid plP421. One copy of another insertion sequence (IS1169) was found 4 bp upstream of the first ATG codon of the nimD gene. This element (1325 bp) shows a strong homology at the nucleotide level (70% identity) with IS 1186 and IS 1168 found to be associated with the Bacteroides carbapenem resistance gene cfiA, and the 5-Nir genes nifiA and nimB, respectively. There is strong evidence that, as in the case of the cfiA gene, the transcription of the four nim genes so far studied is directed by outward-oriented promoters, carried on the right ends of the different insertion sequence elements.

The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign body and wound infections. Recently, the gene encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman was cloned and sequenced. The ClfA protein possesses a highly unusual 308 residue dipeptide repeat region composed predominantly of Asp and Ser. Polymerase chain reaction analysis of seven different strains showed that the size of the clfA repeat coding region varies from 580 bp to 1320 bp. In contrast, the clfA region A is the same size in each strain. The size of the clfA repeat region did not correlate with the ability of these strains to form clumps in a solution of fibrinogen. Indeed, the strain with the smallest repeat size of 580 bp clumped almost as well as strain Newman. Each strain of S. aureus examined contained several high molecular mass proteins that reacted with anti-ClfA region A antibody. In some cases the molecular mass of the major protein varied in accordance with the length of the coding sequence for the repeat region R.

Dichelobacter nodosus is a Gram-negative anaerobic bacterium that is the causative organism of footrot in sheep. A D. nodosus locus responsible for a modification of the host lipopolysaccharide (LPS) in Escherichia coli was cloned and sequenced. Genetic studies showed that the modification occurred within the inner-core region of the host LPS, most likely to one or more of the heptose molecules. Antibodies eluted from the modified LPS reacted preferentially with the lipid-A-core region of D. nodosus LPS, suggesting that the cloned epitope was present in this region of the D. nodosus LPS. The gene responsible for the modification, IpsA, potentially encoded a polypeptide of approximately 37 kDa which was highly basic, a characteristic of enzymes which interact with the acidic inner LPS core. The IpsA gene appeared to be arranged in a complex operon with a downstream gene, prfC, which encoded a protein with similarity to E. coli peptide-chain release factor 3.

Previous publications have demonstrated the presence of a cryptic gene encoding a novel Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Recent Escherichia coli genome sequencing revealed a gene (ptsA) encoding a new Enzyme I homologue in the 89.1-89.3 centisome region. We have analysed this region, and here describe and characterize open reading frames (ORFs) encoding (1) a fused PTS Enzyme I-IIAFru homologue, (2) a glycerol dehydrogenase, (3) a transaldolase homologue, (4) two PTS IIBFru homologues, (5) a PTS IICFru homologue, and (6) homologues of pyruvate formate-lyase and its activating enzyme. Binary comparison scores, multiple alignments and phylogenetic trees establish the families of proteins to which each of the relevant ORFs belong. Identification of the putative products of this gene cluster leads to the proposal that several of the proteins encoded in this region function in anaerobic carbon metabolism.

Pseudomonic acid (mupirocin) produced by Pseudomonas fluorescens is a polyketide antibiotic which blocks isoleucyl-tRNA synthetase. Knowledge of the biosynthetic pathways leading to pseudomonic acid production may help in engineering related antibiotics with other useful properties. To help define these pathways, we have isolated 13 non-producing mutants using Tn5 and Tn1725 mutagenesis. Seven of the Tn5 insertions mapped within a 55 kb region. The remaining insertions, and one gene encoding resistance to pseudomonic acid, mapped at separate locations. DNA that overlapped the seven clustered Tn5 insertions was isolated on a series of clones, extending over a region in excess of 60 kb. Non-producing mutants generated via gene disruption suggest that the biosynthetic cluster extends throughout this region.

Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole. The nucleotide sequence of the C. acetobutylicum DNA controlling metronidazole sensitivity in E. coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated M r of 35000. The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E. coli encoded by the shl gene, and the GaIR, LaCI and PurR repressors of E. coli. The C. acetobutylicum ORF, which was termed regA, complemented a B.subtilis ccpA mutant and an E. coli shl mutant, but was unable to complement E. coli galR, lacI or purR mutants. To determine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading enzyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The RegA protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli.

The dnrF gene, responsible for conversion of aklavinone to ɛ-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050, close to drrAB, one of the anthracycline-resistance genes. The dnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa. The deduced DnrF protein shows significant similarities with bacterial FAD- and NADPH-dependent hydroxylases either required to introduce hydroxyl groups into polycyclic aromatic polyketide antibiotics or involved in catabolism of aromatic compounds. Heterologous expression of dnrF in Streptomyces lividans TK23 and in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield ɛ-rhodomycinone. The enzyme is inactive on anthracyclines glycosylated at position C-7 and its activity decreases to a different extent with other substrate modifications, indicating that DnrF has a significant substrate specificity.

The gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA. Plasmids containing the L. lactis gene were able to complement a gap mutant of Escherichia coli. The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36043. The codon usage in gap and four other glycolytic genes from L. lactis showed a high degree of bias, when compared with 84 other chromosomal genes. Northern blot analysis of total L. lactis RNA showed that gap hybridized strongly with a 1·3 kb transcript. The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon. These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L. lactis gap is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes. The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function. The 3' end of a clpA homologue was identified in the sequence upstream of ORF156. The location of gap on the L. lactis DL11 chromosome map was determined to be between map coordinates 0·530 and 0·660.

The level of diversity, degree of enzyme polymorphism, effective population size, and the relative roles of drift and selection were examined in a cross-section of a natural Escherichia coli population based on random samples of haplotypes of E. coli isolated from sewage. The population studied contained E. coli strains derived from a human population of approximately 16000 individuals, as well as from other sources. Three sample sets were taken between May and August. Each set consisted of 100 E. coli clones. Six enzyme loci [GPI (5 alleles), GPD (5 alleles), PGD (10 alleles), ADH (8 alleles), IDH (6 alleles), PGM (6 alleles)] were surveyed electrophoretically for each clone; 159 different haplotypes were obtained and it is likely that all possible combinations are present in the population sampled. The large numbers of different haplotypes observed were distributed as a series of four genetically similar families of clones. The large estimated effective population size (N e = 1010) means that the observed large and highly significant changes in allele frequencies with time are not due to genetic drift. Selection, though not necessarily at the loci studied, is considered the only likely explanation.