- Volume 139, Issue 2, 1993

The phosphate-irrepressible alkaline phosphatase of the ethanol-producing bacterium Zymomonas mobilis was solubilized from membranes by the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB) and purified by fast protein liquid chromatography. The purified enzyme was a monomeric protein of molecular mass 54 kDa, highly resistant to heat and to ionic strength. The alkaline phosphatase of growing Z. mobilis cells (ZAPase), remained active in the presence of an ethanol concentration as high as 100 g l−1. However, in vitro, the stability of the purified ZAPase was severely affected at low ethanol concentration (7·8 g l−1), showing the importance of the membrane environment in vivo. ZAPase differed from other bacterial alkaline phosphatases by having a higher affinity for the substrate 4-nitrophenylphosphate, a higher K i for phosphate, only a partial reactivation by Zn2+ after EDTA inhibition, and a higher specific activity.

Analytical and preparative isoelectric focusing were used to separate extracellular isoenzymes of aminopeptidase (pI 4.51, M r 45000, pH optimum 7·0) and prolyl-dipeptidylpeptidase (pI 4·01, M r 74000, pH optimum 8·0) produced by the entomopathogenic fungus Metarhizium anisopliae during growth on locust cuticle. Production of both activities is repressed by readily utilized nitrogen sources, but unlike the aminopeptidase, the dipeptidylpeptidase was also excreted at high levels during growth on casein. Casein-grown cultures contained additional isoenzymes with activity against lysyl-alanyl-4-methoxy-2-naphthylamine indicating M. anisopliae possesses multiple peptidases as an adaptation to different nutrient conditions. The aminopeptidase hydrolysed alanyl-leucyl-alanine and showed a broad specificity versus monoaminoacyl β-naphthylamine (βNA) substrates with alanine ßNA being the most rapidly hydrolysed. Inhibition by both bestatin and amastatin indicated similarities to the class of alanyl aminopeptidases (aminopeptidase M). Metal complexing agents also inhibited the aminopeptidase indicating a metal ion requirement. A specific inhibitor for serine proteases [diisopropyl fluorophosphate (DFP)] was without effect. The dipeptidylpeptidase showed a strong preference for substrates having a penultimate proline residue including alanyl-prolyl-glycine and aa-prolyl-βNA substrates. The enzyme showed a broad specificity at the N-terminal amino acid. Inhibition by diprotin A indicates similarities with mammalian prolyl-dipeptidylpeptidases. The enzyme was also inhibited by DFP, implying involvement of a serine residue in catalysis. The results are discussed in the context of cuticle degradation and the participation of exopeptidases as mediators in releasing amino acids necessary for pathogen growth.

The amino acid sequence of the so-called 70 kDa (actually 64 kDa) serine protease secreted by the Gram-negative fish pathogen Aeromonas salmonicida has been determined. It shows a high degree of homology with the complete sequence of other bacterial serine proteases which, with molecular masses of approximately 30 kDa, are less than half its size. This homology is particularly marked in regions adjacent to the catalytic triad Asp32, His64 and Ser221 of subtilisin BPN′. Significant features of the A. salmonicida enzyme, a new member of the group of cysteine-containing subtilisin-type serine proteases, are the presence of six cysteine residues in the mature enzyme, a 37 amino acid extension at the N-terminus and 215 amino acids at the C-terminus when compared with subtilisin BPN'. In addition to a number of smaller peptide insertions there is a non-aligned 32 amino acid sequence in a position corresponding to its introduction between Lys213 and Tyr214 of subtilisin BPN′. This sequence is highly hydrophilic, with Asp/Asn accounting for 10 of the 32 amino acids. Further, the possession of two Cys residues separated by 24 amino acids provides the capacity for stabilizing the peptide as an externalized loop.

Dark-grown resting (non-dividing) cells of Euglena gracilis var. bacillaris and mutants W3BUL (with a proplastid remnant) and W10BSmL (lacking plastids) incubated with 35SO4 2− form a series of labelled lipids which are low or absent in dividing cells. These lipids all release labelled taurine on mild acid-hydrolysis. Treatment of the labelled lipids with 2,4-dinitrofluorobenzene (DNFB) followed by acid hydrolysis does not yield labelled dinitrophenyltaurine (DNP-taurine), but treatment with DNFB after hydrolysis readily forms labelled DNP-taurine, indicating that taurine is linked to the lipids by at least the amino group. Illumination increases the labelling of these taurolipids in plastid-containing cells (wild-type and W3BUL) but has little effect in cells lacking plastids (W10BSmL); labelling is highest in W10 cells irrespective of illumination. This indicates that the presence of a plastid may exert a negative control on taurolipid formation which is relieved by light. The same series of labelled lipids is found in isolated purified mitochondria from mutant W10, indicating that this organelle is a site for taurolipid deposition. The formation of taurolipids under non-dividing conditions may be a response to nutritional stress and these negatively charged constituents (as well as the thylakoid sulpholipid) may serve to protect membranes by repelling deleterious negatively charged oxygen species.

The anchoring (irreversible attachment) of Azospirillium brasilense Cd to hydrophobic polystyrene and to root surfaces was compared. Live A. brasilense Cd cells attached in significantly greater numbers to roots than to polystyrene, regardless of treatments made to the surfaces or to the bacterial cells. Triton X-100, Na2EDTA and several bacterial-inhibitory substances reduced bacterial attachment to both surfaces, although this effect was greater with attachment to polystyrene than to roots. Pre-coating with root exudates, bovine serum albumin or gelatin significantly increased anchoring to both surfaces. Manganese-limited cells showed increased anchoring to roots, whereas dead cells adsorbed better to polystyrene. Although the anchoring of A. brasilense Cd to a non-biological surface can be significantly altered by using several promoting or inhibiting substances to affect the properties of both the surface and the bacterial cell, anchoring to root surfaces is less affected by these substances. It is proposed that at least two different quantitative types of anchoring exist in this bacterium: a sparse attachment to a non-biological surface and a prolific attachment to roots.

Fermentative bacteria were isolated from refuse excavated from municipal solid waste landfills in New York, Florida and Arizona. Anaerobic bacteria cultured on enriched solid media ranged from 105 to 108 c.f.u. (g dry wt of refuse)−1. A significant correlation (P <0·03) was found between numbers of anaerobes cultured at 37 °C and moisture content of refuse. Bacteria hydrolysing starch and protein represented 0-15 % of the total anaerobes cultured; no anaerobic bacteria hydrolysing ball-milled cellulose were isolated. Aerobic bacteria isolated on enriched medium ranged between 104 and 107 c.f.u. (g dry wt)−1. Direct microscopic counts of total bacteria associated with refuse were in the order of 1010 bacteria (g dry wt)−1. These data suggest that, despite relatively high densities of bacteria in landfills, polymer hydrolysis is mediated by a small percentage of the fermentative population.

Lipase of Staphylococcus epidermidis 9 was purified from culture supernatant fluid. Two polypeptides (51 and 43 kDa) were detected by SDS-PAGE, of which the 43 kDa polypeptide reacted with anti-lipase serum. The S. epidermidis 9 lipase gene (gehC) was cloned in Escherichia coli and localized to a 2·1 kb sequence by subcloning and transposon mutagenesis. The nucleotide sequence of gehC (2064 nucleotides) was determined and the predicted amino acid sequence of the encoded lipase (77 kDa) identified. A 97 kDa lipase was detected in extracts of E. coli harbouring gehC and in post-exponential-phase culture supernatant fluids of S. epidermidis 9. Data presented indicate that the lipase behaves anomalously during SDS-PAGE and that a pro-lipase is proteolytically processed in cultures of S. epidermidis 9 during growth.

Sexual cell fusion occurs between NC4 and HM1, heterothallic strains of Dictyostelium discoideum. Several cell surface proteins relevant to the process have been identified. One of them, gp138, exists in fusion-competent cells of both NC4 and HM1, and is considered to be more concerned with membrane fusion than gamete recognition. In this study, we raised monoclonal antibodies against gp138 and examined gp138 distribution among strains and species of cellular slime moulds to confirm its importance in sexual cell fusion. All heterothallic and bisexual D. discoideum strains examined were found to possess gp138, while asexual and homothallic strains lacked it. The anti-gp138 monoclonal antibody detected several distinct proteins in homothallic strains and one in an asexual strain. Some of the former proteins appeared together with the increase in binucleated cells. Cells of Dictyostelium mucoroides and Polysphondylium pallidum did not possess proteins reactive to the monoclonal antibody. These results indicate that gp138 is common among, but restricted to, cross-matable strains of D. discoideum. Our results also support previously published molecular phylogenetic studies which suggest that homothallic and asexual strains of D. discoideum are remote from other strains of D. discoideum but are less distantly related to them than other species are.

Actinomycin D-resistant RNA synthesis was detected in the cellular (VM) fraction which contains large vesicles plus mitochondria from Agrocybe aegerita. It involved the RNAase-resistant complex of naked (unencapsidated) double-stranded RNAs (dsRNAs). The RNA polymerase assay showed [α-32P]UTP incorporation in dsRNAs of a size corresponding to the previously described naked M-dsRNAs and in single-stranded RNAs of related size, which are assumed to act as intermediaries in the replicative cycle of the dsRNA molecules. Moreover, the incorporation of ribonucleotides into large mitochondrial dsDNA fragments was detected in the dsRNA-containing strain. This result favours a mitochondrial location for the naked dsRNAs and their associated polymerizing enzyme(s). No replication of the encapsidated large L-dsRNA viral genome was detected in the VM fraction, supporting the hypothesis that the two types of dsRNAs observed in this A. aegerita strain are independent entities.

Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to a few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg2+-, Mn2+- or Ca2+-dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. calcoaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

The Clostridium thermocellum cell gene, coding for endoglucanase I (Cell), consists of an open reading frame (ORF) of 2640 nucleotides and codes for a protein of M r 98531. The ORF was confirmed as cell by comparing the N-terminal sequence of purified recombinant Cell with that deduced from the nucleotide sequence. Cell hydrolysed lichenan and carboxymethylcellulose, but was principally active against barley-glucan. It exhibited significant sequence identity with subfamily E2 endoglucanases, and by analogy with others in this group contains a catalytic domain of around 500 residues located in the N-terminal half of the protein. The C-terminal region of Cell was highly homologous with the cellulose-binding domain of the non-catalytic cellulosome subunit, S1. A repeated segment, previously shown to be highly conserved in xylanase Z and in other endoglucanases from C. thermocellum, was absent from Cell. Antiserum raised against purified recombinant Cell cross-reacted with proteins contained in the cellulosomes of two strains of C. thermocellum, suggesting that Cell is either a component of the cellulosome or is homologous to other cellulosome proteins. A second gene, located upstream of cell, consisted of an ORF of 1671 nucleotides, coding for a protein of M r 61042. Based on its homology with the Escherichia coli tar gene product, the polypeptide encoded by the second gene is tentatively identified as a sensory transducer.

The DNA sequence of the gene encoding the early and specific immunogenic protein P36 of Mycoplasma hyopneumoniae has been determined. Comparison of the DNA sequence and the deduced amino acid sequence of P36 with known genes and proteins in data banks indicated that P36 is a l-lactate dehydrogenase (LDH) (EC 1.1.1.27). Biochemical analysis of protein P36 expressed from the cloned gene in Escherichia coli confirmed that P36 has l-lactate dehydrogenase activity. Protein P36 of M. hyopneumoniae therefore is termed LDH and its gene Idh. M. hyopneumoniae LDH was shown to containthe typical domains of LDH of other bacterial species. Immunologically however, we have shown that polyclonal antibodies against M. hyopneumoniae LDH do not cross-react with related LDH and show high specificity for M. hyopneumoniae. The Idh gene is preceded by several typical −10 sequences found in promoters of prokaryotes, but lacks the −35 sequence. Sequences rich in A + T, however, precede the −10 boxes, suggesting that factors involved in transcription initiation and their regulation may be different in M. hyopneumoniae compared to other bacterial species, but the putative ribosome binding site seems to be conserved.

A nucleotide sequence encoding an exo-β-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a Bg/II RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.

Candida parapsilosis secretes an inducible acid protease (ACP) when cultivated in the presence of bovine serum albumin as the sole nitrogen source. In order to clone the ACP gene (ACP) of C. parapsilosis, a genomic library was screened with C. tropicalis ACP as the probe. Two different ORFs, ACPR and ACPL, were found to hybridize with the C. tropicalis ACP. ACPRcontained a DNA sequence in agreement with the N-terminal amino acid sequence of C. parapsilosis ACP isolated from culture supernatants. ACPR was shown to be expressed and functional in a C. tropicalis acid protease mutant (acp) and with SDS-PAGE the protein product showed the same mobility as the ACP secreted by C. parapsilosis. These results imply that ACPR encodes the C. parapsilosis ACP. The deduced amino acid sequence of ACPR is similar to the amino acid sequence of proteases of the pepsin family. As in the case of the C. tropicalis and C. albicans ACP, the 5′ extremity of ACPR revealed a propeptide containing two Lys-Arg amino acid pairs that have been identified as peptidase processing sites in several yeast-secreted peptides and protein precursors. As judged from the deducedamino acid sequences, the ACPL product would be similar to that of ACPR; however, a protein corresponding to ACPL was not found in supernatants from C. parapsilosis liquid cultures. In addition, ACPL did not complement the C. tropicalis acp mutant. We conclude that ACPL is a pseudogene or serves an as yet unidentified function.

Expression in the yeast Saccharomyces cerevisiae of the intact nprE gene of Bacillus subtilis, which encodes the pre-pro-NprE neutral protease precursor, resulted in intracellular accumulation of unprocessed precursor without detectable secretion or processing of the expressed gene product. When sequences specifying the signal peptide of yeast invertase were fused upstream of sequences encoding the mature NprE enzyme, nprE gene products were secreted into the culture medium. The secreted protein products were, however, highly glycosylated and biologically inactive.

The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P). The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined. glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP. A σA-type promoter and the transcriptional start point for glpFK were identified. In the 5′ untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found. The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase. Expression of glpFK and glpD requires the inducer G3P and the glpP gene product. A 2·9 kb B. subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7. pLUM7 contains a functional glpP gene as shown by its ability to complement various glpP mutants. Immediately upstream of glpP an open reading frame is found (ORF1). Disrupting ORF1 by plasmid integration in the B. subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source. With the present report all B. subtilis glp genes located at 75° on the chromosomal map have been identified.