- Volume 149, Issue 5, 2003

Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis. APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases. The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins. This study shows that glucose and the activated Ras2Val19 protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast. Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways. Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium. Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function. However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3. Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.

Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active laccase. Relative to the isozymic forms of the native C. subvermispora enzyme, the A. niger-produced laccase had a higher molecular mass and gave a single band on IEF gels. In contrast, A. nidulans transformants secreted several isoforms remarkably similar to those of the native system. Considered together with previously reported Southern blots and protein sequencing, expression in A. nidulans supports the view that C. subvermispora has a single laccase gene and that multiple isoforms result from post-translational processes. In addition, several lines of evidence strongly suggest that under copper limitation, A. nidulans secretes apoprotein which can be reconstituted by a short incubation with Cu(I) and to a lesser extent with Cu(II).

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of l-arabinose. Expression of the three known l-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of l-arabinose or l-arabitol. In addition, the intracellular activities of l-arabitol dehydrogenase and l-arabinose reductase, involved in l-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding d-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. l-Arabitol, most likely the true inducer of the arabinanolytic and l-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the l-arabinose catabolic pathway.

In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5·6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding 55Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.

Co-aggregation among bacterial cells caused by the adherence of one bacterial species to another is a potential colonization mechanism. Several putative aggregation factors for co-aggregation between Porphyromonas (Por.) gingivalis and Prevotella (Pre.) intermedia were partially purified from Por. gingivalis vesicles by gel filtration and affinity chromatography. Antisera against the aggregation factors were made. Analysis using these antisera revealed that 18 and 44 kDa proteins might be responsible for Por. gingivalis vesicle-mediated aggregation of Pre. intermedia. Using antiserum against the 18 kDa protein, the DNA region encoding it was cloned from Por. gingivalis genomic DNA. Sequence analysis revealed that the DNA region was located within the rgpA and kgp genes, encoding Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively, and it encoded non-catalytic adhesin domain regions, namely a C-terminal portion of HGP15, the entire HGP17 sequence and an N-terminal portion of HGP27. A portion of the DNA sequence was also found in the haemagglutinin A (hagA) gene. A recombinant glutathione S-transferase (GST)–HGP17 fusion protein reacted to antiserum against the 18 kDa protein and Pre. intermedia cells could adhere to GST–HGP17-conjugated Sepharose 4B beads, indicating that the HGP17 domain protein is responsible for Por. gingivalis vesicle-mediated aggregation of Pre. intermedia.

Most Escherichia coli strains are resistant to n-hexane. E. coli OST4251 is a n-hexane-sensitive strain that was constructed by transferring the n-hexane-sensitive phenotype from a n-hexane-sensitive strain by P1 transduction. OST4251 is resistant to diphenyl ether, which is less harmful than n-hexane to micro-organisms. The genetic determinant responsible for this subtle difference in the solvent resistance is mapped at 1·2 min on the E. coli chromosome. Nucleotide sequence analysis showed that IS2 and IS5 had integrated upstream of the imp/ostA structural gene in OST4251. The integration of IS2 decreased the activity of the imp/ostA promoter. A product of the gene was identified immunologically as an 87 kDa minor protein associated with the outer membrane. Upon transformation with plasmids containing the imp/ostA gene, OST4251 produced a high level of the gene product in the membrane and acquired n-hexane resistance. Thus, the low level of promoter activity resulted in low Imp production and the n-hexane-sensitivity phenotype. It is likely that the gene product contributes to n-hexane resistance by reducing the influx of n-hexane.

An open reading frame (ORF) that would encode a putative antiterminator protein (LicT) of the BglG family was identified in the genomic DNA sequence of Streptococcus mutans. A DNA sequence that would encode a potential ribonucleic antiterminator (RAT) site in the mRNA at which the putative antitermination protein LicT would bind was located immediately downstream from this ORF. These putative antitermination components are upstream of a glucose-independent β-glucoside-utilization system that is responsible for aesculin utilization by S. mutans NG8 in the presence of glucose. It was hypothesized that these putative regulatory components were an important mechanism that was involved with the controlled expression of the S. mutans bglP locus. A strain of S. mutans containing a licT : : Ω-Kan2 insertional mutation was created. This strain could not hydrolyse aesculin in the presence of glucose. The transcriptional activity associated with other genes from the bgl regulon was determined in the licT : : Ω-Kan2 genetic background using lacZ transcriptional fusions and β-galactosidase assays to determine the effect of LicT on these loci. The LicT protein had no significant effect on the expression of the bglC promoter, a regulator of the bglA locus. However, it is essential for the optimal expression of bglP. These data correlate with the phenotype observed on aesculin plates for the S. mutans wild-type strain NG8 and the licT : : Ω-Kan2 strain. Thus, the glucose-independent β-glucoside-specific phosphotransferase system (PTS) regulon in S. mutans relies on LicT for BglP expression and, in turn, aesculin transport in the presence of glucose. Interestingly, LicT also seems to negatively regulate the expression of the bglA promoter region. In addition, the presence of the S. mutans licT gene has been shown to be able to activate a cryptic β-glucoside-specific operon found in Escherichia coli.

The mshB gene encoding N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5–10 % of the parent strain mc2155) but did not abolish mycothiol synthesis completely. Complementation of the MshB− mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB− mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB− mutant did not exhibit increased resistance to isoniazid. However, the MshB− mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA− mutants also exhibit ethionamide resistance but that a MshC−mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.

Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N2 fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains of Escherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogen E. coli O157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuated E. coli K-12 strain. In the case of stable mutators, mutS defects in strains identified among natural isolates were analysed, including two E. coli O157 : H7 strains, a diarrhoeagenic E. coli O55 : H7 strain, and a uropathogenic strain from the E. coli reference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient in mutS transcripts or produced transcripts truncated at the 3′ end. Nucleotide sequence analysis revealed extensive deletions in the mutS region of three strains, ranging from 7·5 to 17·3 kb relative to E. coli K-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in the mutS coding sequence. These results provide insights into the status of the mutS gene and its product in pathogenic strains of E. coli.

This study demonstrates that Escherichia coli insertion elements IS3, IS150 and IS186 are able to form transpositionally active head-to-tail dimers which show similar structure and transpositional activity to the dimers of IS2, IS21 and IS30. These structures arise by joining of the left and right inverted repeats (IRs) of two elements. The resulting junction includes a spacer region (SR) of a few base pairs derived from the flanking sequence of one of the reacting IRs. Head-to-tail dimers of IS3, IS150 and IS186 are unstable due to their transpositional activity. They can be resolved in two ways that seem to form a general rule for those elements reported to form dimers. One way is a site-specific process (dimer dissolution) which is accompanied by the loss of one IS copy along with the SR. The other is ‘classical’ transposition where the joined ends integrate into the target DNA. In intramolecular transposition this often gives rise to deletion formation, whereas in intermolecular transposition it gives rise to replicon fusion. The results presented for IS3, IS150 and IS186 are in accordance with the IS dimer model, which is in turn consistent with models based on covalently closed minicircles.

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique ‘hypothetical’ protein (PA0572), which could not be detected in the culture supernatants of Δlas mutants, although they were unaffected in Δrhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

pSV1 is a plasmid in Streptomyces violaceoruber SANK95570 that carries the methylenomycin biosynthetic (mmy) gene cluster. An ordered cosmid map and an EcoRI map have been constructed for pSV1, confirming that pSV1 is a 163 kb circular plasmid. The mmy gene cluster has been found on three different replicon structures; the circular plasmid pSV1, the 356 kb linear plasmid SCP1 and, via SCP1 integration, the linear chromosome of Streptomyces coelicolor A3(2). Comparison of pSV1 and SCP1 sequences revealed that the two plasmids have homology to each other only around the mmy and parAB regions, eliminating models in which pSV1 was generated by circularization of SCP1 or vice versa. It is likely that the mmy gene cluster was horizontally transferred as a set together with the parAB region in the comparatively recent evolutionary past.

Whether micro-organisms can live in periapical endodontic lesions of asymptomatic teeth is under debate. The aim of the present study was to visualize and identify micro-organisms within periapical lesions directly, using fluorescence in situ hybridization (FISH) in combination with epifluorescence and confocal laser scanning microscopy (CLSM). Thirty-nine periapical lesions were surgically removed, fixed, embedded in cold polymerizing resin and sectioned. The probe EUB 338, specific for the domain Bacteria, was used together with a number of species-specific16S rRNA-directed oligonucleotide probes to identify bacteria. To control non-specific binding of EUB 338, probe NON 338 was used. Alternatively, DAPI (4′,6′-diamidino-2-phenylindole) staining was applied to record prokaryotic and eukaryotic DNA in the specimens. Hybridization with NON 338 gave no signals despite background fluorescence of the tissue. The eubacterial probe showed bacteria of different morphotypes in 50 % of the lesions. Rods, spirochaetes and cocci were spread out in areas of the tissue while other parts seemed bacteria-free. Bacteria were also seen to co-aggregate inside the tissue, forming microcolonies. Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis and treponemes of phylogenetic Group I were detected with specific probes. In addition, colonies with Streptococcus spp. were seen in some lesions. A number of morphotypes occurred that could not be identified with the specific probes used, indicating the presence of additional bacterial species. CLSM confirmed that bacteria were located in different layers of the tissue. Accordingly, the FISH technique demonstrated mixed consortia of bacteria consisting of rods, spirochaetes and cocci in asymptomatic periapical lesions of root-filled teeth.