- Volume 149, Issue 4, 2003

The C and D cell cycle periods of seven Escherichia coli K-12 strains and three E. coli B/r strains were determined by computer simulation of DNA histograms obtained by flow cytometry of batch cultures grown at several different generation times. To obtain longer generation times two of the K-12 strains were cultivated at several different dilution rates in glucose-limited chemostats. The replication period (C period) was found to be similar in K-12 and B/r strains grown at similar generation times. At generation times below 60 min the C period was constant; above 60 min it increased linearly with increasing generation time. The period from termination of replication to cell division (D period) was more variable. It was much shorter in B/r than in K-12 strains. Like the C period it was relatively constant at generation times below 60 min and it increased with increasing generation times at longer generation times. In glucose-limited chemostats good correlation was found between D periods and generation times, whereas batch cultures exhibited carbon-source-dependent variations. Chemostat cultures showed cell cycle variations very similar to those obtained in batch cultures. These flow cytometric determinations of cell cycle periods confirm earlier determinations of the C period and establish that the D period also varies with generation time in slowly growing cultures. In addition they extend the range of growth rates at which cell cycle periods have been determined in E. coli K-12.

Burkholderia cepacia isolates of genomovar III are highly transmissible amongst patients with cystic fibrosis (CF) and express a 97 kDa putative haem-binding protein (HBP) [ Smalley, J. W., Charalabous, P., Birss, A. J. & Hart, C. A. (2001) . Clin Diagn Lab Immunol 8, 509–514]. An investigation of the interactions of iron(III) protoporphyrin IX with epidemic and non-epidemic strains of B. cepacia to determine the role of the above protein in haem acquisition and binding is reported herein. Spectrophotometric titrations of cell suspensions of genomovar IIIa strains BC7 and C5424 with iron(III) protoporphyrin IX, at pH 7·0, resulted in the depletion of Fe(III)PPIX.OH monomers and formation of the μ-oxo oligomeric species, [Fe(III)PPIX]2O. Difference spectroscopy indicated a continuous conversion of the monomeric iron(III) protoporphyrin IX into μ-oxo oligomers. Incubations with Fe(III)PPIX.OH monomers at pH 6·5 also showed that cells could shift the equilibrium to generate the μ-oxo oligomeric form. Genomovar I strains ATCC 25416 and LMG 17997 were unable to mediate this conversion. SDS-PAGE of genomovar IIIa strains exposed to Fe(III)PPIX.OH at pH 6·5 followed by tetramethylbenzidine/H2O2 staining revealed, in addition to the 97 kDa HBP, two proteins of 77 and 149 kDa located in the outer membrane which bound Fe(III)PPIX.OH monomers. These proteins were absent from the genomovar I strains. Genomovar IIIa strains BC7 and C5424 showed increased cellular binding of [Fe(III)PPIX]2O, and as a consequence, displayed increased catalase activities compared to cells of the genomovar I isolates. It is concluded that, in addition to the putative 97 kDa HBP, B. cepacia genomovar IIIa strains express two outer-membrane proteins which function to bind and convert Fe(III)PPIX.OH monomers into the μ-oxo oligomeric form, [Fe(III)PPIX]2O. The ability to perform this conversion at both neutral and slightly acidic pHs may enable epidemic strains to withstand attack from neutrophil-derived H2O2 in the inflamed CF lung.

Natural transformation is a mechanism for intra- and interspecific transfer of chromosomal DNA in Pseudomonas stutzeri. During this process a single strand derived from duplex DNA is transported into the cytoplasm and recombined with resident DNA. By electroporation, which introduces duplex DNA into cells, 100-fold lower transformation frequencies of P. stutzeri JM300 were observed with shuttle vector or broad-host-range plasmid DNA when the plasmids had replicated in Escherichia coli and not in P. stutzeri JM300. Moreover, the natural transformation with cloned chromosomal P. stutzeri JM300 DNA was reduced about 40-fold when the DNA had not been propagated in P. stutzeri JM300 but in E. coli. Restriction was also active during natural transformation by single-stranded DNA. Restriction during natural transformation and electroporation was abolished in mutants isolated from mutagenized JM300 cells after applying a multiple plasmid electroporation strategy for the enrichment of restriction-defective strains. The mutants had retained the ability for DNA modification. The P. stutzeri strain ATCC 17587 was found to have no restriction–modification system as seen in JM300. It is discussed whether restriction during natural transformation acts at presynaptic or postsynaptic stages of transforming DNA. Restriction as a barrier to transformation apparently contributes to sexual isolation and therefore may promote speciation in the highly diverse species P. stutzeri.

The tetrachlorobenzene dioxygenase (TecA) of Ralstonia sp. PS12 carries out the first step in the aerobic biodegradation of chlorinated toluenes. Besides dioxygenation of the aromatic ring of 4-chloro-, 2,4-, 2,5- and 3,4-dichlorotoluene as the main reaction, it also catalyses mono-oxygenation of the methyl groups of 2,3-, 2,6-, 3,5-di- and 2,4,5-trichlorotoluene as the main reactions, channelling these compounds into dead-end pathways. Based on the crystal structure of the homologous naphthalene dioxygenase (NDO) and alignment of the α-subunits of NDO and TecA, the substrate pocket of TecA was modelled. Recently, for NDO and the homologous 2-nitrotoluene dioxygenase (2NTDO), two amino acids (Phe352 of NDO and Asn258 of 2NTDO) were identified which control the regioselectivity of these enzymes. The corresponding amino acids at Phe366 and Leu272 of TecA were substituted to change the regioselectivity and to expand the product spectrum. Position 366 was shown to control regioselectivity of the enzyme, although mutations resulted in decreased or lost activity. Amino acid substitutions at Leu272 had little or no effect on the regioselectivity of TecA, but had significant effects on the product formation rate. Substitutions at both positions changed the site of oxidation of 2,4,5-trichlorotoluene slightly. As new products, 3,4,6-trichloro-1-methyl-1,2-dihydroxy-1,2-dihydrocyclohexan-3,5-diene, 4,6-dichloro-3-methylcatechol, 3,6-dichloro-4-methylcatechol and 3,4-dichloro-6-methylcatechol were identified.

Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients. One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells. The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates. The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster. This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface. Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm. In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit. These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis. It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B. cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l−1 were shown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.

The CobT enzyme of Salmonella typhimurium was shown in vitro to have NAD+-dependent ADPribosyltransferase activity. The CobT enzyme transferred the ADPribosyl moiety of NAD+ onto 5,6-dimethylbenzimidazole (DMB) yielding a new dinucleotide, namely α-5,6-dimethylbenzimidazole adenine dinucleotide (α-DAD), whose identity was established by mass spectrometry. The N 1-(α-d-ribosyl)-5,6-dimethylbenzimidazoyl moiety (α-ribazole) of α-DAD was incorporated into adenosylcobalamin (AdoCbl) by cell-free extracts of S. typhimurium, indicating that α-DAD served as an intermediate of AdoCbl biosynthesis. The rate of transfer of the ADPribosyl moiety was slower than the rate of transfer of the phosphoribosyl moiety of nicotinate mononucleotide (NaMN) to DMB. The CobT enzyme displayed a low K m for NaMN (0·51 mM) relative to the one for NAD+ (9 mM); nicotinate adenine dinucleotide (NaAD) and nicotinamide mononucleotide (NMN) also served as substrates for CobT. In spite of the high K m of CobT for NAD+, the latter is proposed to be a relevant physiological substrate of CobT, given that the intracellular concentrations of NaMN, NMN and NaAD in actively growing S. typhimurium are undetectable. Evidence shows that extracts of S. typhimurium contain an as-yet unidentified dinucleotide pyrophosphatase that can cleave α-DAD into α-ribazole-5′-P and AMP; α-ribazole-5′-P can then enter the AdoCbl biosynthetic pathway.

Pseudomonas putida strain DS1 utilizes dimethyl sulfide (DMS) as a sulfur source, and desulfurizes it via dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO2) and methanesulfonate (MSA). Its Tn5 mutant, Dfi74J, no longer utilized DMS, DMSO and DMSO2, but could oxidize DMS to DMSO2, suggesting that the conversion of DMSO2 to MSA was interrupted in the mutant. Sequencing of the Tn5 flanking region of Dfi74J demonstrated that a gene, sfnR (designated for dimethyl sulfone utilization), encoding a transcriptional regulator containing an ATP-dependent σ54-association domain and a DNA-binding domain, was disrupted. sfnR is part of an operon with two other genes, sfnE and sfnC, located immediately upstream of sfnR and in the same orientation. The genes encode NADH-dependent FMN reductase (SfnE) and FMNH2-dependent monooxygenase (SfnC). Complementation of Dfi74J with an sfnR-expressing plasmid led to restoration of its growth on DMS, DMSO and DMSO2. An rpoN-defective mutant of strain DS1, which lacks the σ 54 factor, grew on MSA, but not on DMS, DMSO and DMSO2, indicating that SfnR controls expression of gene(s) involved in DMSO2 metabolism by interaction with σ 54-RNA polymerase. Northern hybridization and a reporter gene assay with an sfn–lacZ transcriptional fusion elucidated that expression of the sfnECR operon was induced under sulfate limitation and was dependent on a LysR-type transcriptional regulator, CysB. This is believed to be the first report that a σ 54-dependent transcriptional regulator induced under sulfate limitation is involved in sulfur assimilation.

Here, the codon usage patterns of two Clostridium species (Clostridium perfringens and Clostridium acetobutylicum) are reported. These prokaryotes are characterized by a strong mutational bias towards A+T, a striking excess of coding sequences and purine-rich leading strands of replication, strong GC-skews and a high frequency of genomic rearrangements. As expected, it was found that the mutational bias dominates codon usage but there is some variation of synonymous codon choices among genes in the two species. This variation was investigated using a multivariate statistical approach. In the two species, two major trends were detected. One was related to the location of the sequences in the leading or lagging strand of replication, and the other was associated with the preferential use of putatively translational optimal codons in heavily expressed genes. Analyses of the estimated number of synonymous and non-synonymous substitutions among orthologous genes permit us to postulate that optimal codons might be selected not only for speed but also for accuracy during translation.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30–54 % and 50–57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn2+-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

The important opportunistic pathogen Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium and a member of the normal resident human gastrointestinal microbiota. Our earlier studies indicated that there is considerable within-strain variation in polysaccharide expression, as detected by mAb labelling. Analysis of the genome sequence has revealed multiple invertible DNA regions, designated fragilis invertible (fin) regions, seven of which are upstream of polysaccharide biosynthesis loci and are approximately 226 bp in size. Using orientation-specific PCR primers and sequence analysis with populations enriched for one antigenic type, two of these invertible regions were assigned to heteropolymeric polysaccharides with different sizes of repeating units, as determined by PAGE pattern. The implication of these findings is that inversion of the fin regions switches biosynthesis of these polysaccharides off and on. The invertible regions are bound by inverted repeats of 30 or 32 bp with striking similarity to the Salmonella typhimurium H flagellar antigen inversion cross-over (hix) recombination sites of the invertible hin region. It has been demonstrated that a plasmid-encoded Hin invertase homologue (FinB), present in B. fragilis NCTC 9343, binds specifically to the invertible regions and the recombination sites have been designated as fragilis inversion cross-over (fix) sites.

A new genetic locus was identified in Rhodobacter sphaeroides which is required for optimal synthesis of the light-harvesting spectral complexes as well as for optimal growth under anaerobic conditions with dimethyl sulfoxide (DMSO) as a terminal electron acceptor. The primary structure of the deduced osp gene product shows significant homology to the receiver domain of known response regulators common to bacterial two-component systems. However, site-directed mutagenesis revealed that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway. Paradoxically, the effect of the Osp protein upon spectral complex levels is exerted at the transcriptional level of photosynthesis gene expression. The absence of the Osp protein does not appear to have a general effect on house-keeping metabolism. In cells lacking Osp, the levels of DMSO reductase appear to be normal. The quaternary structure of the Osp protein was determined to be a homodimer and it was directly demonstrated that Osp does not bind to the promoter region of photosynthesis genes as judged by mobility-shift experiments and primary structure analysis.

Ribonucleotide reductases (RNRs) are a family of complex enzymes that play an essential role in all organisms because they catalyse de novo synthesis of deoxyribonucleotides required for DNA replication and repair. Three different classes of RNR have been described according to their metal cofactors and organic radicals. Class Ib RNR is encoded in four different genes (nrdH, nrdI, nrdE and nrdF) organized in an operon. The authors previously cloned and sequenced the genes encoding the active class Ib RNR of Corynebacterium ammoniagenes and showed that these genes are clustered in an atypical nrdEF region, which differs from that of other known class Ib enzymes because of an intergenic sequence (1171 bp) present between nrdE and nrdF. This study investigated the transcriptional organization and regulation of this nrd region by RT-PCR. Three different and independent mRNA were found (nrdHIE, nrdF and an ORF present in the intergenic region), each one being transcribed from its own promoter and being essential for normal growth. The ratio of nrdF to nrdHIE mRNA was 9·1, as determined by competitive RT-PCR; the expression of both nrdHIE and nrdF was found to be dependent on the culture growth phase, and was induced in the presence of hydroxyurea, manganese and hydrogen peroxide. This is believed to be the first direct evidence for a manganese-dependent transcriptional regulation of nrd genes. These results suggest a common and coordinated regulation of the different nrd genes, despite their being transcribed from independent promoters.

Clostridium thermocellum produces one major β-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237T (6848 bp) and F7 (9766 bp). Overlapping sequences were 99·9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-β-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, β-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-β-glucanase with a specific activity of 2680 and 340 U mg−1 and a K m of 0·94 and 2·1 mg ml−1 towards barley β-glucan and laminarin, respectively. It was specific for β-glucans containing β-1,3-linkages with an optimum temperature of 70 °C at pH 6·0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on β-1,3- or β-1,3-1,4-glucan.

Twenty-five genes are involved in methanol oxidation to formaldehyde by the methanol dehydrogenase system in the facultative methylotroph Methylobacterium extorquens AM1 organized in five gene clusters. RT-PCR was used to assess the transcripts for the main gene clusters that encode methanol dehydrogenase and proteins required for its activity (mxaFGJIRSACKLDEHB), and the enzymes that are required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone (pqqABC/DE and the pqqFG cluster). In both cases, positive bands were obtained corresponding to mRNA spanning each of the genes in the cluster, but not across the first and last genes and the gene immediately upstream or downstream of the cluster, respectively. These results suggest that these three gene clusters are each transcribed as a single operon. Confirmation was obtained by cloning a number of intergenic regions into a promoter probe vector. None of these regions showed significant promoter activity. Promoter regions were analysed for mxaF, pqqA, orf181 upstream of pqqFG, and mxaW, a gene located upstream of mxaF and divergently transcribed. The promoter regions for these genes were defined to within 100, 46, 124 and 146 bp, respectively, and the two unknown transcriptional start sites were determined, for mxaW and orf181. Alignment of these promoter regions suggests that they all may be transcribed by the σ 70 orthologue in M. extorquens AM1.