Exoenzyme Production Research Articles

Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production.

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

Virulence determinants in Pseudomonas aeruginosa strains from urinary tract infections.

A total of 121 uropathogenic Pseudomonas aeruginosa strains were examined for production of several virulence-related factors. These strains were distributed in five predominant O-serotypes, i.e. O 4, O 12, O 11, O 6 and O 5, which accounted respectively for 23.9, 23.1, 12.3, 8.2 and 5.7% of isolates. Pyochelin and pyoverdin siderophores were produced by most of the isolates, defective variants occurring at very low frequency (2.4% for pyochelin and 7.4% for pyoverdin). Adherence to uroepithelial cells and production of cytotoxins was demonstrated in 52.8 and 67.7% of the strains, respectively, with higher frequencies for epidemiologically related strains belonging to serotypes O 4 and O 12. Titration of total proteases, elastase and phospholipase C revealed a high degree of heterogeneity among isolates. However, examination of individual O-serotypes by exoenzyme production showed that elevated levels of total proteases and elastase were characteristics of serotypes of minor numerical importance, i.e. O 1, O 10, O 11 and O 17, whilst low levels of elastase were produced by strains belonging to the predominant serotypes, namely O 4 and O 12. Moreover, epidemiologically related strains belonging to serotypes O 4 and O 12 appeared more homogeneous than the whole serogroup, when compared with other groups on the basis of exoenzyme levels.

Failure of phenotypic characteristics to distinguish between carrier and invasive isolates of Staphylococcus epidermidis

Thirty carrier and 29 invasive Staphylococcus epidermidis isolates were analysed for production of slime, extracellular enzymes and antibiotic resistance. Evaluation of slime production was by two methods which gave differing results, but both showed no difference between the two groups of isolates. Exoenzyme production by the two groups of isolates was also similar, and the only differences were shown in resistance to some antibiotics; carrier isolates were more resistant to novobiocin and cephalothin, but resistance to chloramphenicol, tunicamycin, teicoplanin, penicillin, ampicillin and tetracycline was similar in both groups. Cluster analysis based on exoenzyme production showed no consistent difference between invasive and carrier isolates, with the lowest similarity coefficient over 88% (except for one isolate). Consequently, none of the characteristics studied was useful in differentiating potentially invasive isolates.

Effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes: a study on mycelial and wall-less phenotypes of the fz; sg; os-1 ('slime') triple mutant of Neurospora crassa

The production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of Neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain RCP-3) of a cross 'slime' x wild type. The unrelated stable 'slime' strain FGSC 1118 was also studied. The synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-catabolite repression for both mycelium-forming phenotypes (mycelial intermediate and spheroplast-hyphal intermediate) of strain RCP-3. The stable 'slime' from RCP-3 produced cellulose-degrading activity and xylan-degrading activity constitutively but was fully sensitive to glucose repression. The stable 'slime' RCP-3 did not synthesize polygalacturonic-acid-degrading activity, even in the presence of inducers. For the stable 'slime' FGSC 1118, all of the polysaccharide-degrading activities were produced constitutively and were markedly resistant to glucose repression. The possible epigenetic origin of the different properties of stable 'slimes' RCP-3 and FGSC 1118 is considered. These results may relate to the role of the cell surface in the processing of regulatory signals which control the adaptation of the fungal cell to the nutritional environment.

Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi, and three other coagulase-negative staphylococci in a mouse model and possible virulence factors

Staphylococcus lugdunesis and Staphylococcus schleiferi, two newly described species, have been isolated from numerous types of human infections. We compared the pathogenicity of 30 strains of S. lugdunensis, S. schleiferi, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus hominis, using a mouse model in which a foreign body preadhered with the test strain was implanted subcutaneously, followed by injection of the test strain. All five species of staphylococci produced abscesses. Staphylococcus epidermidis, S. schleiferi, and S. lugdunensis yielded species means of 76-91% abscess formation; 80-100% of the infected foreign bodies and tissues were culture positive. These three species were more virulent than S. warneri or S. hominis, which produced abscesses in 54 and 65% of mice, respectively; only 10-48% of the infected samples were culture positive. Transmission electron microscopy of pure cultures of selected strains showed that all species possessed glycocalyx. All species produced a variety of possible virulence factors, such as alpha and delta hemolysins, as well as the aggressins lipase and esterase. The production of exoenzymes did not always correlate with virulence as demonstrated by abscess formation in mice.

Phenotypic conversion of Pseudomonas aeruginosa in cystic fibrosis

Pseudomonas aeruginosa strains isolated from cystic fibrosis patients were tested for production of exoenzymes, sensitivity to pooled normal human serum, and colony morphology. Strains isolated from patients exhibiting a severe form of the disease were seen to produce a decreased range of exoenzymes, to show an increase in their serum sensitivity, and to be predominantly mucoid in colonial character compared with strains isolated from patients with a milder form of the disease. These results suggest that P. aeruginosa undergoes phenotypic changes with respect to exoenzyme secretion, serum sensitivity, and colony form as the clinical condition of the cystic fibrosis patient changes.

Effect of environmental phosphate concentration on protein export by Bacillus licheniformis NCIB 6346

Bacillus licheniformis NCIB 6346 was grown under phosphate or magnesium limitation in continuous culture and forward and reverse transitions carried out to examine their effects upon extracellular enzyme production. Analysis of the observed kinetics allowed a distinction to be made between the effects of phenotypic variation and of genetic selection upon exoenzyme production. Furthermore, increases in the phosphate concentration (0.3–30 mM) were directly related to increased phenotypic expression of alpha-amylase. By contrast, penicillinase production was reduced under these conditions but analysis revealed that its phenotypic expression was only indirectly influenced by the concentrations of either magnesium or phosphate present during the transitions.

Lipase production of Staphylococcus carnosus in a dialysis fermentor

In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.

Bacterial Physiological Diversity in the Rhizosphere of Range Plants in Response to Retorted Shale Stress

Bacterial populations were isolated from the soil-root interface and root-free regions of Agropyron smithii Rydb. and Atriplex canescens (Pursh) Nutt. grown in soil, retorted shale, or soil over shale. Bacteria isolated from retorted shale exhibited a wider range of tolerance to alkalinity and salinity and decreased growth on amino acid substrates compared with bacteria from soil and soil-over-shale environments. Exoenzyme production was only slightly affected by growth medium treatment. Viable bacterial populations were higher in the rhizosphere and rhizoplane of plants grown in retorted shale than in plants grown in soil or soil over shale. In addition, a greater number of physiological groups of rhizosphere bacteria was observed in retorted shale compared with soil alone. Two patterns of community similarity were observed in comparisons of bacteria from soil over shale with those from soil and retorted-shale environments. Root-associated populations from soil over shale had a higher proportion of physiological groups in common with those from the soil control than with those from the retorted-shale treatment. However, in non-rhizosphere populations, bacterial groups from soil over shale more closely resembled the physiological groups from retorted shale.

Hyperproduction of an intracellular heterologous protein in a sacUh mutant of Bacillus subtilis

The sacR regulatory region which controls inducible expression of sacB, the gene for extracellular levansucrase, was isolated on a 500-bp fragment of Bacillus subtilis chromosomal DNA. The region was separated into two components: a 325-bp fragment which carries a constitutive promoter and a 175-bp fragment which carries a stem-loop structure presumably involved in the induction process. The constitutive sacR promoter was used to drive expression of the plasmid-borne xylE gene, coding for intracellular catechol 2,3-dioxygenase (C23O), with its Pseudomonas ribosome-binding site, in otherwise isogenic sacU −, sacU + and sacU h mutant strains of B. subtilis. Mutations at the sacU locus have been previously shown to affect multiple cell functions, particularly production of extracellular enzymes. The presence of the sacU h mutation allowed for hyperproduction of C23O to levels exceeding 25% of total cellular protein. This represents a 50- to 100-fold enhancement over levels observed in sacU − and sacU + host cells. A 5- to 10-fold improvement of C23O production in sacU h cells was observed when the subtilisin ( aprA) promoter was used in place of sacR. In contrast, a bacteriophage T5 synthetic promoter was found to be independent of host strain for high-level synthesis of C23O. Hyperproduction of an intracellular protein in sacU h cells suggests that enhancement of exoenzyme production, previously observed in these mutants, occurs prior to the secretion event. Therefore, hyperproduction is most likely due to elevated transcription or translation or specific nucleotide sequences.

Simple screening method for identification of nonpleiotropic mutants for exoenzyme production.

A differential medium that distinguishes between pleiotropic and nonpleiotropic mutants for exoenzyme production has been developed for Staphylococcus simulans biovar staphylolyticus. The medium will facilitate genetic analysis of exoenzyme production by this organism. Generally useful strategies for increasing the sensitivity of indicator plates for detection of exoenzyme activities are presented.

Biotyping and exoenzyme profiling as an aid in the differentiation of human from bovine group G streptococci.

Group G streptococci were isolated from throat and extrapharyngeal cultures from 75 patients during an 18-month period. Of 29 throat isolates, 18 were recovered from patients with pharyngitis, 8 were of unknown significance, and 3 were of questionable etiology. Clinical significance could be ascribed to 13 of 46 extrapharyngeal isolates recovered from wound, urinary tract, blood, and conjunctival cultures. Extrapharyngeal isolates recovered from stool, sputum, and vaginal cultures were considered nonsignificant. A total of 96 group G streptococcal strains (including 21 human and 14 bovine strains from outside sources) were tested for exoenzyme production and subjected to a large battery of biochemical tests. Bovine and human isolates could be distinguished on the basis of trehalose fermentation, litmus milk reduction, and production of beta-D-glucuronidase, hyaluronidase, and fibrinolysin. Eight distinct biotypes could be discerned on the basis of fermentation of trehalose, raffinose, and lactose and esculin hydrolysis. All isolates that fermented raffinose were associated with infection. These results support the concept of two distinctly different epidemiological reservoirs of group G streptococci in humans and bovines.

Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S.

Exoenzyme S is an extracellular product of Pseudomonas aeruginosa. This enzyme catalyzes the transfer of ADP-ribose from NAD to a number of as yet unidentified eucaryotic proteins, but it is distinct from toxin A. To evaluate the role of exoenzyme S in the pathogenicity of P. aeruginosa, we isolated transposon-induced mutants of strain 388, a clinical isolate that produces exoenzyme S but no toxin A. The transposon Tn1 was introduced by using a temperature-sensitive derivative of plasmid RP1. A Tn1-induced mutant was found which had no detectable exoenzyme S activity or antigen in culture supernatants or in cell lysates. Except for its lack of exoenzyme S and resistance to carbenicillin, this mutant was indistinguishable from the parent strain. When tested in an experimental mouse burn infection model, this Tn1-induced mutant was reduced in virulence by at least 2,000-fold, suggesting a role for exoenzyme S in the virulence of this strain.

Effects of amino acids and derivatives of cyclic adenosine 3′, 5′-monophosphate on the production of three exoenzymes by [formula omitted] biovar [formula omitted

The extracellular protease, endopeptidase, and hexosaminidase produced by Staphylococcus, simulans biovar staphylolyticus were neither induced nor repressed by amino acids but required a tryptic digest of casein for their production. Catabolite repression of exoenzyme production by glucose was not affected by exogenous cyclic adenosine 3′, 5′-monophosphate but was partially relieved by di- or monobutyryl derivatives of this compound.

Classification of Chronic Blepharitis

Since last thoroughly evaluated over three decades ago, the clinical spectrum of chronic blepharitis has changed. The relative prevalence of Staphylococcus aureus alone or in combination with seborrheic blepharitis has decreased. The relative prevalence has increased of seborrheic blepharitis with or without associated excess meibomian secretions (meibomian seborrhea) or inflammation (meibomitis). Primary meibomitis appears not to be a primarily infectious entity but to represent a facet of generalized sebaceous gland dysfunction and to be found in association with seborrheic dermatitis or acne rosacea. The keratoconjunctivitis found in association with primary meibomitis may be contributed to by the production of bacterial lypolytic exoenzymes that split neutral lipids, resulting in an increased level of free fatty acids in the tears. A frequent finding of keratoconjunctivitis sicca in this patient population, especially the S. aureus group (50%), is of note. Of particular importance is that these entities be recognized as chronic diseases requiring control and ones for which there is no “cure.”

Exoenzymes of Propionibacterium acnes.

Thirty strains of Propionibacterium acnes were grown in basal salt medium containing lecithin as a lipid substrate and in other media. The cultures were assayed for production of lipase (measured as fatty acid esterase) and other exoenzymes. Lipase was assayed spectrophotometrically; other enzymes were assayed using the API ZYM system (Analytab Products Inc., Plainview, NY). Substance for lipase were alpha- and beta-naphthol esters of propionic, butyric, valeric, caprylic, lauric, myristic, and oleic acids. All strains showed fatty acid esterase activity. Using the API ZYM system 19 enzymes were detected, 8 of which were found frequently and had high activity in most strains. Acid and alkaline phosphatases, phosphoamidase, ester lipase, trypsin-chymotrypsin-like proteases, beta-glucuronidase (80%), beta-galactosidase (80%), and N-acetyl-beta-glucosaminidase were found. Many enzymes of P. acnes appear to be adaptive, dependent on the culture substrate.

Production of exoenzyme S by clinical isolates of Pseudomonas aeruginosa.

Exoenzyme S differs from toxin A and diphtheria toxin in that it does not adenosine diphosphate (ADP)-ribosylate elongation factor-2, but rather catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide to a number of different proteins in extracts of eucaryotic cells. Polyoma-transformed BHK-21 cells were isolated which were resistant to diphtheria toxin and toxin A. Extracts from these cells are ADP-ribosylated by exoenzyme S but not toxin A or diphtheria toxin, providing an assay which distinguishes between S and A activities. A total of 124 clinical isolates of P. aeruginosa were analyzed for production of toxin A and exoenzyme S. Exoenzyme S production was detected in 38% of the strains, whereas 80% of the strains produced toxin A.

Catabolite‐Like Repression of Extracellular Enzyme Production in Vibrio parahaemolyticus

Production of extracellular amylase and protease in Vibrio parahaemolyticus was repressed by various carbohydrates present in the medium. In addition, the protease production was repressed very strongly by peptones or casamino acids. Cyclic adenosine 3', 5'-monophosphate (cyclic AMP) added exogenously could reverse the repression of amylase production, but not that of protease production irrespective of the "repressors" used. Mutants of V. parahaemolyticus, which resembled the reported cya (adenylate cyclase) and crp (cyclic AMP receptor protein) mutants of Escherichia coli and related organisms, were examined for the exoenzyme production. Amylase production in the mutants was defective, while their protease production was not defective, but rather accentuated as compared with that in the parental strain. These findings strongly suggest that amylase production is subject to catabolite repression mediated by cyclic AMP, whereas protease production is controlled by a repression mechanism which mimics in part, but may be distinct from catabolite repression.

Preferential suppression of normal exoenzyme formation by membrane-modifying agents.

Cerulenin, phenethyl alcohol, benzyl alcohol, procaine, and a series of aliphatic alcohols selectively suppressed production of active exoenzymes by various bacterial strains.

Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.