The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

TolC mutant of Sinorhizobium meliloti strain SKHM1-188 fails to establish effective symbiosis with alfalfa

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain SKhM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix- nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.     

The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage

The TolC protein of Escherichia coli is implicated in a variety of diverse cellular functions, including antibiotic efflux and alpha-hemolysin secretion. An incidental role of TolC is to facilitate the entry of the bacteriophage TLS and colicin E1 into the bacterial cell. Despite the resolution of TolC's atomic structure, the roles of specific residues in its diverse functions are unknown. Here, we describe a genetic strategy for isolating missense tolC mutations that abolish the bacteriophage receptor activity of the TolC protein without influencing its role in antibiotic efflux. These spontaneous mutations affected two regions of the TolC protein and included base-pair substitutions, insertions, and deletions. Comparison of the TolC sequence with those of its homologues revealed two hypervariable stretches that were predicted to represent loops. Interestingly, all but one of the TolC alterations preventing phage binding were located in these two hypervariable regions, which are likely to be exposed on the cell surface. This was substantiated by the recently solved three-dimensional structure of TolC. Curiously, all the phage-resistant TolC mutants showed varying degrees of resistance to colicin E1, suggesting the involvement of overlapping regions of TolC in colicin E1 import and phage binding.The phage used in this study, TLS, was earlier reported as a strain of U3. However, we show here that, unlike the previously reported lipopolysaccharide-specific U3 phage, this phage displays a distinctly different host range and discrete morphological features and, in addition to utilizing TolC as receptor, it requires the inner core of a lipopolysaccharide.     

MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.     

Mutant invertase proteins accumulate in the yeast endoplasmic reticulum

Summary Intercompartmental transport of secreted proteins in yeast was analysed using invertase mutants. Deletions and insertions at the BamHI (position +787) or the Asp718 (position +1159) sites of the SUC2 gene led to mutant proteins with different behaviour regarding secretion, localization and enzyme activity. The deletion mutants showed accumulation of core glycosylated material in the endoplasmic reticulum (ER) a decrease of secreted protein by 5%–30% and loss of enzyme activity. The secreted material was localized in the culture medium and not — as is normal for invertase-in the cell wall. No delay in transport from the Golgi to the cell surface was observed, indicating that the rate-limiting step for secretion is at the ER-Golgi stage. Two insertion mutants, pIPA and pIPB, retained enzyme activity. Mutant pIPB showed 10% secretion, while 60%–70% secretion was observed for pIPA. While the non-secreted material accumulated in the ER, the secreted material was present in the cell wall. The results suggest that the presence of structures incompatible with secretion leads to ER accumulation of mutated invertase.     

<Emphasis Type="Italic">tolC</Emphasis> mutant of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> strain CXM1-188 fails to establish effective symbiosis with alfalfa

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain CXM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing Calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix^? nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.     

Expression and biochemical characterization of the periplasmic domain of bacterial outer membrane porin TdeA

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.     

Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis

The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain reduced secretion to the extracellular space. Many of these mutants were undetectable, but secretion of a subset of mutants with insertions in the N-terminal half of the toxin could be restored to wild type secretion levels if cultured in the presence of 0.1% Triton X-100. Secretion of two mutants with insertions at the extreme C terminus was partially restored when co-expressed with a minimal clone of EspP, a related autotransporter protein. Several well secreted mutants with insertions in the N-terminal third of the molecule reduced protease activity over 20-fold, suggesting that the protease domain is located within this N-terminal region of Pet. We have also identified two insertional mutants in the middle of the passenger domain that were proteolytic but no longer cytopathic; these mutants displayed decreased binding and internalization upon incubation with HEp-2 cells. Our data suggest the existence of separate functional domains mediating Pet proteolysis, secretion, and cell interaction.     

The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.     

Genetic Analysis of the Colicin V Secretion Pathway

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.     

Role of the carboxy-terminal region of the outer membrane protein AatA in the export of dispersin from enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. AatA, an outer-membrane protein that is a homolog of E. coli TolC, facilitates the export of the dispersin protein Aap across the outer membrane in EAEC. To identify which amino acids are important for this export activity, site-directed mutagenesis of the carboxy terminus was performed. An insertional mutant of aatA was complemented with each of several deletion mutants, and was examined for Aap secretion. The results showed that three nonpolar amino acids at positions 381-383 (Phe-Leu-Leu) were required for the activity, and these residues were located at the base of carboxy-terminal elongation in the equatorial domain of AatA.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

The ttsA gene is required for low-calcium-induced type III secretion of Yop proteins and virulence of Yersinia enterocolitica W22703

Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain.     

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ～70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.     

Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC . In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca²⁺ (Cl) for their growth at 37°C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD₅₀ for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH—LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.     

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.     

Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA(218)) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10(4) E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23 degrees C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA(218) fusion protein at 23 and 30 degrees C but unexpectedly not at 37 degrees C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA(218) was detected only in the L448F mutant (AE104F) at 23 degrees C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA(218) fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37 degrees C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.     

Colony morphology mutants of Trichoderma harzianum with increased beta-1,4-N-acetyl-glucosaminidase production

A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive beta-1,4-N-acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.