- Volume 140, Issue 7, 1994

This paper presents and compares the assimilation rates of CO2 and lactate, and the lactate respiration rates, of attached bacterial populations growing in slowly flowing groundwater (1-3 mm s-1) from deep crystalline bed-rock of the Stripa research mine, Sweden. The bacteria studied grew in anoxic, high-pH (9-10) and low-redox artesian groundwater flowing up through tubing from two levels of a borehole designated V2, 812-820 m and 970-1240 m below ground. Bacteria were allowed to attach to and grow on sterile glass microscope slides in laminar-flow reactors connected to the flowing groundwater. Total numbers of bacteria were counted by acridine orange direct counts. The bacteria grew slowly, with doubling times of 34 d at 10 °C for the 812-820 m population, 23 d for the 970-1240 m population at 10 °C and 16 d for this population at 20 °C. Numbers of attached bacteria reached between 106 and 107 bacteria cm-2. The populations at the two levels of the borehole were different in physiology as well as in phylogeny and reflected the heterogeneity between the sampling levels. The earlier proposed presence of sulphate-reducing bacteria could not be confirmed. This is discussed in relation to results from 16S rRNA gene sequencing studies. The CO2 assimilation rates (as mol CO2 cm-2 h-1, using liquid scintillation techniques) increased with depth and temperature. The quotients calculated for inorganic/organic carbon utilization were between 0·07 and 0·25, indicating that autotrophy could not support the levels of growth observed and that heterotrophy was the dominant carbon transformation process for growth of the studied populations. The Stripa bacteria could further be seen not only to assimilate but also to catabolize lactate and release CO2, which added to the indications of a heterotrophic dominance in the Stripa environment.

This paper presents the molecular characterization of attached bacterial populations growing in slowly flowing artesian groundwater from deep crystalline bed-rock of the Stripa mine, south central Sweden. Bacteria grew on glass slides in laminar flow reactors connected to the anoxic groundwater flowing up through tubing from two levels of a borehole, 812-820 m and 970-1240 m. The glass slides were collected, the bacterial DNA was extracted and the 16S rRNA genes were amplified by PCR using primers matching universally conserved positions 519-536 and 1392-1405. The resulting PCR fragments were subsequently cloned and sequenced. The sequences were compared with each other and with 16S rRNA gene sequences in the EMBL database. Three major groups of bacteria were found. Signature bases placed the clones in the appropriate systematic groups. All belonged to the proteobacterial groups beta and gamma. One group was found only at the 812-820 m level, where it constituted 63% of the sequenced clones, whereas the second group existed almost exclusively at the 970-1240 m level, where it constituted 83% of the sequenced clones. The third group was equally distributed between the levels. A few other bacteria were also found. None of the 16S rRNA genes from the dominant bacteria showed more than 88% similarity to any of the others, and none of them resembled anything in the database by more than 96%. Temperature did not seem to have any effect on species composition at the deeper level. SEM images showed rods appearing in microcolonies. The conditions at the levels differ in pH, temperature, redox and flow rate, and in content of sulphate, iron and sulphide. The presence of one dominant species in the laminar-flow reactors at each level indicates that the environments might have offered restrictive physical or physiological conditions difficult to adapt to.

We have cloned a Candida albicans gene, which encodes a cyclic nucleotide phosphodiesterase (PDEase), by complementation in a Saccharomyces cerevisiae PDEase-deficient mutant. The deduced amino acid sequence is similar to that of the low-affinity PDEase of S. cerevisiae (PDE1) and the cyclic nucleotide PDEase (PD) of Dictyostelium discoideum. Biochemical analysis of recombinant protein produced in S. cerevisiae indicated that the enzyme behaves as a PDE1 homologue: it hydrolyses both cAMP (K m = 0·49 mM) and cGMP (K m = 0·25 mM), does not require divalent cations for maximal activity and is only moderately inhibited by millimolar concentrations of standard PDEase inhibitors. Based on these data, we designate the C. albicans we have cloned, PDE1. Low-stringency genomic Southern blots showed cross-hybridization between C. albicans PDE1 and DNA from Candida stellatoidea, but not with DNA from S. cerevisiae or several closely related Candida species.

The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the α polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the α polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli β polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.

The position of junctions and the extent of the duplicated chromosomal regions in Bacillus subtilis merodiploid strains were studied by quantitative DNA-DNA hybridization. We describe a method which allows (i) the identification of genes present in two copies per chromosome and (ii) the measurement of the amount of additional DNA in chromosomes with relatively large duplicated regions (about 10% or more). Analysis of previously described B. subtilis merodiploid strains GSY1127, GSY1800 and GSY1835 revealed that the duplicated segments represent 29 ± 2%, 7 ± 2% and 13 ± 2% of the chromosome, respectively. Small discrepancies between these and previous genetic linkage data are discussed. Support for a role of prophage SPβ in the formation of merodiploid GSY1835 is provided. In conclusion, the described method confirmed the genetic maps of the merodiploids previously obtained by transduction and transformation crosses and showed that a duplication of a segment is not accompanied by large deletions of other chromosomal regions, providing direct evidence that a cell can accommodate genomes of substantially increased size.

Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample containing 1 μg DNA and 107 exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10-5, as compared to 10-4 routinely obtained by the method of transformation in liquid medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell density, medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiological conditions.

The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 16S, 23S and 5S rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 16S rRNA and 23S rRNA genes and the spacer-2 region separating the 23S rRNA and 5S rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, ‘Mycobacterium lufu’ and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 5S rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase III processing sites within the spacer-1 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 23S rRNA and for precursor 5S rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 5S rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species.

Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 °C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, M r 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M r 132000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350000; B form, 600000; A and B forms after endoglycosidase H (endo H) treatment, 180 000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M r centred at ~ 100 000 (A) and ~ 150 000 (B) but were reduced to a single 58 000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of M r ~ 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both p-nitrophenylGIcNAc (pNPGIcNAc) and pNPGalNAc at pH 4·0. The kinetic parameters k cat (s-1) and K m (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGIcNAc, 740, 0·77; pNPGalNAc, 910, 1·26; N,N'-diacetylchitobiose 620, 0·20; and N,N',N-triacetylchitotriose, 170, 0·044. The enzyme showed substrate inhibition with all substrates above 0·5 mM except for pNPGalNAc.

The composition of lipopolysaccharide (LPS) extracted with aqueous phenol from a virulent English plum isolate of Pseudomonas syringae pv. morsprunorum varied according to the partial pressure of oxygen (pO2) in the culture medium at the time of harvest. When pO2 was low, the organism grew slowly and produced smooth LPS bearing rhamnan sidechains. As pO2 was raised, the rate of growth increased and smooth LPS was replaced by a rough species deficient in rhamnose, which co-extracted with a d-glucan. Organization of rhamnose and glucose into separate polymers was shown by the selective susceptibility of the rhamnose-containing polymer to hydrolysis by rhamnanase of the phage A7. By methylation analysis, GC-MS, and 1H- and 13C-NMR spectroscopy, the glucan was shown to consist of α(1 → 4)-linked residues with α(1 → 4,6)-branch points and non-reducing terminal residues in the approximate ratio 4:1:1, resembling glycogen in composition. A glucan which co-extracted with LPS using phenol/water from an avirulent plum isolate that was resistant to lysis by phages A1 and A7 was shown by methylation analysis to have a similar structure. Whether the effect on LPS composition was due directly to pO2, or was dependent on the rate of growth, has not been established. It is suggested that, because epiphytic growth would entail exposure to high pO2, English plum isolates growing on the surfaces of host plants might be unable to produce smooth LPS. Since cell surface composition affects virulence in plant-pathogenic pseudomonads, this effect could account for the observed failure of the English plum isolates to enter the host via leaf scars.

Weak and strong light/heat stimuli induced changes in cyclic guanosine monophosphate (cGMP) levels in vegetative and aggregation competent amoebae and in slug cells of the Dictyostelium discoideum strain X22. Mutant strains derived from X22 with mutations in the phototaxis loci phoA-phoK fell into four phenotypic classes with respect to cGMP responses to weak and strong light/heat stimuli. These results suggest an intermediary role for cGMP in photosensory and thermosensory processing in slugs and amoebae. The streamer F mutant NP368 which has previously been shown to exhibit a prolonged cGMP response to cAMP, showed a wild-type cGMP response to light and heat. All phototaxis mutant strains with altered cGMP responses to light and heat were unaltered in their cGMP response to cAMP. These results suggest that cAMP and light/heat regulate cGMP via independent pathways. The thermotaxis mutant HPF228 showed altered cGMP responses to heat but not to light stimuli. This suggests that the mutation in HPF228 affects thermosensory transduction before convergence with the phototaxis pathway and the subsequent cGMP response.

During a 6 h incubation at 26 °C, Mucor hiemalis hydroxylated progesterone predominantly at the 14α site. Further incubation resulted in the production of several 14α-dihydroxyprogesterones and a novel microbial transformation product, 8(9), 14(15)-didehydroprogesterone (pregna-4,8[9], 14[15]-triene-3,20-dione). Azole inhibition indicates that the hydroxylases are cytochromes P-450. Mycelia transformed in the presence of the translation inhibitor cycloheximide failed to hydroxylate progesterone, whereas identical treatment with the transcription inhibitor actinomycin D, or heat-shock for 1 h at 37 °C prior to progesterone transformation, stimulated hydroxylation. 14α-Hydroxylation was stimulated if mycelia were pre-incubated with progesterone, transcription or translation inhibitors. These data are consistent with a model in which cytochrome P-450 steroid hydroxylase mRNA is stored in cells in a dormant form by a labile sequestering protein.

To examine pathways of glutamate and glutamine metabolism in the ectomycorrhizal fungus Paxillus involutus, tracer kinetic experiments were performed using l-[U-14C]glutamate and L-[U-14C]glutamine and the enzyme inhibitors methionine sulfoximine (MSX), azaserine (AZA) and aminooxyacetate (AOA). When [14C]glutamate was supplied to fungal cultures, 25% of the radioactivity of the amino acid fraction was incorporated into glutamine after 5 min feeding, but MSX inhibited incorporation of 14C into glutamine by 85%, suggesting the rapid operation of glutamine synthetase. Conversely, when P. involutus was fed with [14C]glutamine, 46% of the label was found in glutamate within 30 min of feeding and AZA inhibited glutamate formation by 90%. Taken together, these data indicate that glutamate synthase (GOGAT) is the major enzyme of glutamine degradation. In addition, the strong inhibition of glutamine utilization by AOA indicates that glutamine catabolism in P. involutus might involve a transamination process as an alternative pathway to GOGAT for glutamine degradation. The high 14CO2 evolution shows that glutamate and glutamine are further actively consumed as respiratory substrates, being channelled through the tricarboxylic acid (TCA) cycle and oxidized as CO2. It appears that synthesis of amino acid precursors during TCA cycle operation is an essential step for aspartate and alanine synthesis through aminotransferase activities in P. involutus.

The high concentrations of short-chain soluble polyphosphates, previously reported in Phytophthora palmivora, have been found in six other species of Phytophthora, and appear to be characteristic of this genus. The distribution of phosphorus among the various acid-soluble pools was similar in all species except P. infestans, which accumulated less polyphosphate than other species when grown in high phosphate medium, and contained several unique and as yet unidentified organic phosphorus compounds. When grown in the presence of potassium phosphonate sufficient to reduce growth by 50%, all species showed an increased accumulation of phosphorus in both pyrophosphate and polyphosphate, without parallel increases in sugar phosphate or nucleotide phosphorus pools. The internal concentration of phosphonate required to produce 50% inhibition varied with species, ranging from 4·6 to 52 μmol (g dry wt)-1. Assimilation of orthophosphate was only slightly reduced in the presence of phosphonate, resulting in an increased concentration of phosphorus per unit mass. Metalaxyl, which also inhibits growth of Phytophthora spp. did not cause accumulation of either pyro- or polyphosphate. Phosphonate treatment also resulted in the formation of a compound identified as isohypophosphate, a metabolite of phosphonate not previously reported in Phytophthora. Taken together, these observations suggest that the primary site of phosphonate inhibition in Phytophthora spp. lies in the metabolism of pyrophosphate.

A cytosolic trehalase was purified from the thermophilic fungus Humicola grisea var. thermoidea. The apparent molecular mass of the purified native enzyme was estimated to be 360 kDa by gel filtration chromatography. A single polypeptide band of approximate molecular mass 120 kDa was detected by SDS-PAGE, suggesting that the native enzyme was composed of three identical polypeptides. The carbohydrate content of the enzyme was estimated to be 12%. The pl of the enzyme, estimated by electrofocusing, was about 4·0. The purified trehalase was specific for trehalose and its activity was stimulated by manganese, calcium and cobalt and inhibited by aluminium chloride, copper sulfate, ADP and ATP. Sugars such as cellobiose, lactose, sucrose, fructose and maltose also showed some inhibitory effect which was abolished in the presence of calcium. The purified cytosolic trehalase exhibited an apparent K m of 0·86 mM, a pH optimum of 5·5 and an optimum temperature of 60 °C. Treatment with calcium induced disaggregation of the enzyme into three components. Apparently, calcium-induced disaggregation was not a prerequisite for calcium-mediated activation of the enzyme. The disaggregated trehalase was more labile to temperature than the native enzyme. The properties of the two H. grisea trehalases: the cytosolic enzyme and the conidial one, were compared.

An NADPH-dependent L-xylulose reductase [xylitol:NADP 4-oxidoreductase; EC 1·1.1·10 (LXDH)] from Aspergillus niger was purified and characterized. It is an octamer with a monomeric molecular mass of 32 kDa, showing high specificity for L-xylulose, xylitol and NADP(H). The K m values for L-xylulose and xylitol are relatively high (16·5 and 925 mM, respectively). An NAD+-dependent xylitol dehydrogenase [xylitol:NAD+ 2-oxidoreductase; EC 1·1.1·9 (DXDH)] was partially purified from the same fungus. It has a monomeric molecular mass of 40 kDa and shows a high specificity for D-xylulose, xylitol and NAD(H). The K m values for D-xylulose and xylitol are relatively low (approximately 4 and 50 mM, respectively). The reactivity towards xylitol, the product or substrate these enzymes have in common, confirms their role in the L-arabinose catabolic pathway.