Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings.

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.

Molecular analysis of a bacterial chitinolytic community in an upland pasture.

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.

Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax.

Several brominated tyrosine derived compounds, psammaplins A (1), K (2) and L (3) as well as bisaprasin (4) were isolated from the Fijian marine sponge Aplysinella rhax during a bioassay guided isolation protocol. Their structures were determined using NMR and MS techniques. Psammaplin A was found to moderately inhibit chitinase B from Serratia marcescens, the mode of inhibition being non-competitive. Crystallographic studies suggest that a disordered psammaplin A molecule is bound near the active site. Interestingly, psammaplin A was found to be a potent antifungal agent.

Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene.

The chitinase of Autographa californica nucleopolyhedrovirus (AcMNPV) is required for the characteristic liquefaction of baculovirus-infected insect larvae. Alignments of the putative active sites of a range of chitinases revealed two highly conserved residues, glutamate and aspartate, which have been proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses were generated, AcchiA(D311G), AcchiA(E315G) and AcchiA(D311G E315G), which contained mutations at either the glutamate, the aspartate or both. It was demonstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the attenuation of chitinolytic activity and the cessation of liquefaction of Trichoplusia ni larvae infected with AcchiA(D311G E315G). Mutagenesis of the glutamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, AcchiA(E315G), liquefied more slowly than those larvae infected with wild-type AcMNPV. Mutagenesis of the aspartate residue resulted in a reduction of exochitinase activity but an unexpected enhancement of endochitinolytic activity. Liquefaction of AcchiA(D311G)-infected larvae was observed at the same time as that of AcMNPV-infected larvae.

Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis.

Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains. These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V. Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis. One gene (PbrCHS3) was not expressed at detectable levels. The others were regulated by morphology and/or by the growth phase of the organism. Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells. This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis.

RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis.

Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America. The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated. Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders. These results underline the belief that P. brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment.

Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase.

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited Km(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with kcat = 1150 min-1. The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with nH = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1. 21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

Aggressive and defensive roles for chitinases.

Chitinases are produced by a wide variety of pathogenic and parasitic microbes and invertebrates during their attack on chitin-containing organisms. Examples discussed include enzymes of insect and algal viruses, of yeast killer toxin plasmids, of bacterial and fungal pathogens of fungi and insects, and of parasitic protozoa. These chitinases play roles in penetration of fungal cell walls, and of exoskeletons and peritrophic membranes of arthropods. Salivas of some invertebrate predators have chitinolytic activity which may be involved in their attack on their prey. Chitinases play a major defensive role in all plants against attack by fungi, and perhaps also against attack by insect pests. The plant chitinases form a very large and diverse assemblage of enzymes from two families of glycosyl hydrolases. At least some vertebrates, including fish and humans, also may utilise chitinases in their defence against pathogenic fungi and some parasites.

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis.

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.

Regulation of chitin synthesis during dimorphic growth of Candida albicans.

Candida albicans has three genes encoding chitin synthase enzymes. In wild-type strains, the expression of CHS2 and CHS3 peaked 1-2 h after the induction of hyphal growth, whilst mRNA levels in a non-germinative strain, CA2, remained low under the same conditions. CHS1 gene expression did not peak during germ tube formation but remained at low levels in both yeast and hyphal growth. The pattern of gene expression did not predict the changes in measured chitin synthase activities or changes in chitin content during dimorphic transition. Chitin synthase activity increased steadily, and did not peak shortly after germ tube induction, and activity profiles were similar in germ-tube-competent and germ-tube-negative strains. The phenotype of a delta chs2 null mutant suggested that CHS2 encoded the major enzyme activity in vitro and was largely responsible for elevated chitin synthase activities in microsomal preparations from hyphal cells compared to yeast cells. However, CaChs3p was responsible for synthesis of most chitin in both yeast and hyphae. Three independent chitin assays gave markedly different estimates of the relative chitin content of yeast and hyphae and wild-type and chs mutants. Only one of the methods gave a significantly higher chitin content for hyphal compared to yeast cell walls and a lower chitin content for hyphae of the delta chs2 null mutant compared to the parental strain.

Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment.

The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.

Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes.

We examined the role of the Autographa californica nucleopolyhedrovirus (AcMNPV)-encoded chitinase in virus pathogenesis in Trichoplusia ni larvae. In conjunction with the AcMNPV-encoded cathepsin, it promotes liquefaction of the host in the latter stages of infection. Insects infected with virus mutants lacking either the chitinase A gene (chiA) or cathepsin gene (cath) remained intact several days after death. However, if both viruses were used to infect insects, liquefaction of the host was restored. Chitinase was readily detected in AcMNPV-infected insects using a chitinase-specific antibody, but it was absent from insects infected with a chiA deletion mutant (AcchiA-). The chitinase was also detected in polyhedra purified from AcMNPV-infected insects but not in those from AcchiA-. However, polyhedra derived from a virus lacking an intact chiA were no less effective in initiating an infection in second instar T. ni larvae than those of the unmodified AcMNPV. It was also demonstrated that the virus chitinase retained high levels of activity between pH 3.0 and 10.0. In contrast, chitinases isolated from Serratia marcescens, although active under acidic conditions, rapidly lost activity above pH 7.0 illustrating that despite 57% sequence identity, the two proteins have distinct enzymic activities.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure.

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2-3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 micrograms/ml; cantharidin, 20 microM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 micrograms/ml; H-7, 100 microM) and a Ca2+ channel blocker (verapamil, 50 microM) reduced chitin synthase activity to 50-60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.

Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase.

The Candida albicans ADH1 gene encodes an alcohol dehydrogenase which is immunogenic during infections in humans. The ADH1 gene was isolated and sequenced, and the 5'- and 3'-ends of its mRNA were mapped. The gene encodes a 350 amino acid polypeptide with strong homology (70.5-85.2% identity) to alcohol dehydrogenases from Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. The cloned C. albicans ADH1 gene was shown to be functional through complementation of adh mutations and efficient production of active alcohol dehydrogenase in S. cerevisiae. Northern analysis of C. albicans RNA revealed that ADH1 mRNA levels were regulated in response to carbon source and during batch growth. During growth on glucose, ADH1 mRNA levels rose to maximum levels during late exponential growth phase and declined to low levels in stationary phase. The ADH1 mRNA was relatively abundant during growth on galactose, glycerol, pyruvate, lactate or succinate, and less abundant during growth on glucose or ethanol. Alcohol dehydrogenase levels did not correlate closely with ADH1 mRNA levels under the growth conditions studied, suggesting either that this locus is controlled at both transcriptional and post-transcriptional levels, or that other differentially regulated ADH loci exist in C. albicans.

Inhibition of chitinolytic activities from tree species and associated fungi.

Effects of two inhibitors, allosamidin and (2-acetamido-2-deoxy-D-glucopyranosylidene)amino phenylcarbamate (PUGNAC), have been assessed on chitinolytic activities of two plants, Pinus sylvestris L. and Eucalyptus pilularis Sm., and of seven fungi. Pinus sylvestris and E. pilularis root endochitinase activities were inhibited by allosamidin. Activities of P. sylvestris were more sensitive to inhibition than those of E. pilularis. The mechanism of inhibition varied with the plant species and the enzyme involved. PUGNAC inhibited beta-N-acetylglucosaminidase and exochitinase activities in root extracts from both plant species. In all cases PUGNAC acted as a reversible competitive inhibitor. Both inhibitors also affected chitinolytic activities from the fungi screened. Allosamidin inhibited endochitinase activities from both the mycorrhizal and pathogenic fungi tested. In addition, exochitinase activity from the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. was inhibited by allosamidin. PUGNAC inhibited beta-N-acetylglucosaminidase activity from all the fungi tested. PUGNAC was also a potent inhibitor of both exo- and endochitinase activities from the fungi, except P. involutus. Competitive inhibition was the most common form. These findings show allosamidin does inhibit endochitinase activity in plants and the ability of PUGNAC to inhibit not only beta-N-acetylglucosaminidase activity but also fungal endochitinase activity may be useful to distinguish between host and fungal endochitinase activities in symbiotic or pathogenic dual systems.

Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans.

Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the cDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strong homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans delta hsp90/delta hsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans.

Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.

A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.

Structure and regulation of a Candida albicans RP10 gene which encodes an immunogenic protein homologous to Saccharomyces cerevisiae ribosomal protein 10.

The Candida albicans clone cDNA10 was isolated on the basis that it encodes a protein which is immunogenic during infections in humans (R. K. Swoboda, G. Bertram, H. Hollander, D. Greenspan, J. S. Greenspan, N. A. R. Gow, G. W. Gooday, and A. J. P. Brown, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45 degrees C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.