- Volume 143, Issue 5, 1997

This work reports the role of both superoxide dismutases - CuZnSOD (encoded by SOD1) and MnSOD (encoded by SOD2) - in the build-up of tolerance to ethanol during growth of Saccharomyces cerevisiae from exponential to post-diauxic phase. Both enzyme activities increase from the exponential phase to the diauxic shift and from the diauxic shift to the post-diauxic phase. The levels of mRNA-SOD1 and mRNA-SOD2 increase from the exponential phase to the diauxic shift; however, during the post-diauxic phase mRNA-SOD1 levels decrease while mRNA-SOD2 levels remain unchanged. These data indicate the existence of two regulatory mechanisms involved in the induction of SOD activity during growth: synthesis de novo of the proteins (until the diauxic shift), and post-transcriptional or post-translational regulation (during the post-diauxic phase). Ethanol does not alter the activities of either enzyme in cells from the diauxic shift or post-diauxic phases, although the respective mRNA levels decrease in post-diauxic-phase cells treated with ethanol (14% or 20%). Results of experiments with sod1 and sod2 mutants show that MnSOD, but not CuZnSOD, is essential for ethanol tolerance of diauxic-shift and post-diauxic-phase cells. Evidence that ethanol toxicity is correlated with the production of reactive oxygen species in the mitochondria is obtained from results with respiration-deficient mutants. In these cells, the induction of superoxide dismutase activity by ethanol is low; also, the respiratory deficiency restores the capacity of sod2 cells to acquire ethanol tolerance.

Escherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0⋅ 5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv. viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem. The V max of cytochrome c reduction fell from 7.1 s-1in the presence of 0⋅5 mM CO to 5⋅0 s-1in the presence of 100 μM O2with no significant change in Km for cytochrome c (6⋅8 to 7⋅3 μM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 μM O2 Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2 electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered.

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component sytems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.

The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at −50.5 and −94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR*(FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR*repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR*protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo. The α-subunit of RNAP interacted with two regions (centred at −105 and −46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at −50.5 prevents the α-subunit of RNAP from docking with the DNA just upstream of the −35 element. Binding of a second FNR*molecule at the −105 site likewise prevents binding of the α-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the α-subunit of RNAP.

We have previously described a new Escherichia coli K-12 mutant, LH530, which has a defective outer membrane. LH530 is very sensitive to hydrophobic antibiotics, does not grow at 42 ° and synthesizes reduced amounts of lipid A. Phenotypically LH530 is very similar to the known lipid A biosynthesis mutants of E. coli and Salmonella typhimurium. Its genetic defect is not known, but the defect is suppressed by multiple copies of ORF195. Here we show that at 37 ° LH530 contains a reduced amount of the OmpF porin and that it leaks periplasmic °-lactamase at 37 °. and 42 °. We further show that ORF195, when present at low copy number, restores the antibiotic resistance and lipid A biosynthesis of LH530 at 28 °, but not at higher temperatures. In contrast, OmpF expression is restored at 37 °.

The cloning and molecular genetic analysis of a locus mapping within the flagellar gene (fli) complex of Salmonella typhimurium is reported. A copy of the insertion element IS200 was located in a noncoding stretch of DNA upstream of the fliA gene. Comparative nucleotide sequence analysis showed that this copy of IS200 was 711 bp long and that its flanking regions contained no features common to other characterized insertion sites of this element. The element was located 37 bp downstream of an ORF whose product was shown by interspecific transfer and amino acid analysis to carry out N-methylation of selected lysine residues in Salmonella flagellin. The sequence and phenotype of this ORF identified it as fliB, encoding the only prokaryotic N-methylase acting on amino groups to have been characterized to date. It was found to be conserved among all clinically significant serovars of Salmonella. The IS200 insertion site is of particular interest since it was conserved in all but two rare evolutionary lines of S. typhimurium, and was absent from 85 Salmonella strains belonging to 37 other serovars. It is thus a phylogenetically significant marker at the serovar level.

A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.

A cluster of flagellar genes of Borrelia burgdorferi was identified and sequenced. This cluster comprises an operon, designated the flgK operon, which is initiated by a s70-like promoter. The flgK operon consists of flbF (function unknown), flgK (encoding HAP1), flgL (encoding HAP3) and orfX (function unknown), and maps at 185 kb on the chromosome. In other bacteria, the hook-associated proteins HAP1 and HAP3 connect the flagellar filament to the hook and are required for the last stage of flagellar assembly. Reverse transcriptase-PCR analysis indicated that flbF through to orfX are transcribed as a single mRNA, and primer extension analysis revealed that transcription of the flgK operon is initiated by a s70-like promoter upstream of flbF. Subcloning the flgK promoter element into a promoter probe cat vector revealed that the flgK promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. In addition, when this construct was transformed into a fliA mutant of S. typhimurium which lacked a functional flagellar-specific s28factor, the flgK promoter was still functional. Based on these results, the promoter element of the flagellin gene (fla, hereafter referred to as flaB) was re-examined. flaB encodes the flagellar filament protein, and a sgp33-34-like promoter has been reported to be involved in the transcription of this gene. A transcriptional start point was found 1 bp downstream of the reported start site. The sequence around -10 and -35 are consistent with the presence of a s70-like promoter in addition to the putative sgp33-34-like promoter for flaB. In contrast to the flgK promoter element, no activity was detected after subcloning a flaB promoter element into the promoter probe cat vector. Because a s70-like promoter rather than a unique flagellar sigma factor is involved in the later stage of flagellar assembly, the regulation of B. burgdorferi flagellar genes is evidently different from that of other bacteria.

A new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 16 kb DNA fragment was sequenced and an ORF of 954 bp identified. Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (Mpcll) from Alcaligenes eutrophus JMP 222. Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage. The enzyme required Fe2+for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.

The nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-β -glucanase lamA (laminarinase) from Thermotoga neapolitana, was determined. It contains an ORF encoding a protein of 646 aa (73 328 Da). The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-1,3-β -D-glucanases and belongs to family 16 of glycosyl hydrolases. This domain is flanked on both sides by one copy on each side of a substrate binding domain homologue (family II). The recombinant laminarinase protein was purified from Escherichia coli host cells in two forms, a 73 kDa and a processed 52 kDa protein, both having high specific activity towards laminarin (3100 and 2600 U mg-1, respectively) and K m values of 2.8 and 2.2 mg ml-1, respectively. Limited activity on 1,3-1,4-β -glucan (lichenan) was detected (90 U mg-1). Laminarin was degraded in an endoglucanase modus, yielding glucose, laminaribiose and -triose as end products. Thus lamA classifies as an endo-1,3(4)-β -glucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95° (73 kDa) and 85° (52 kDa) at an optimum pH of 6.2. The superior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100°, where 57% of the initial activity remained after 30 min (82% at 95°). Thus, lamA is the most thermostable 1,3-β -glucanase described to date.

Immunological screening of a Pseudomonas aeruginosa cosmid library led to the identification of clones producing an 18 kDa outer-membrane protein. This protein reacted in Western blots with a polyclonal antiserum against outer-membrane proteins of P. aeruginosa and with a monoclonal antibody (MA1-6) specific for OprL, the peptidoglycan-associated outer-membrane lipoprotein (PAL). Sequencing of pOML7, a subclone expressing oprL, revealed an ORF of 504 bp encoding a polypeptide with a typical lipoprotein signal recognition sequence. Another ORF was found upstream of oprL, with homology to the ToIB protein of Escherichia coli and Haemophilus influenzae. Downstream of oprL, a second ORF, of 321 bp, was found (orf2), encoding a protein with a signal peptide and with no homology with proteins of known biological function. After the stop codon of orf2, a rho-independent terminator sequence was detected which is part of the P. aeruginosa PA01 insertion element IS222. OprL showed homologies with all known PALs from Gram-negative bacteria, especially in the C-terminal part. mAb MA1-6 reacted with P. aeruginosa cells in immunofluorescence, and with E. coli cells expressing oprL, which had an abnormal, elongated morphology, an indication that production of the protein perturbed the division process.

Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as ‘phylogenetic stains’ in the identification of bacteria. In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, Paraphysomonas vestita. The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique P. vestita sequences 18-21 nucleotides in length. Five sequences in different parts of the SSU rRNA gene were used to design 5 -fluorescently labelled oligonucleotide probes. Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent. All five probes targeted to P. vestita proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria. These probes provide a means of quantitatively measuring the proportion of P. vestita cells in samples of mixed protists.