Nonmucoid Variants Research Articles

Phenotypic variation in exopolysaccharide production in the marine, aerobic nitrogen-fixing unicellular cyanobacterium Cyanothece sp.

The aerobic nitrogen-fixing cyanobacterium, Cyanothece sp. BH68K produces non-mucoid variants defective in exopolysaccharide (EPS) production at a high frequency. The EPS-producing wild-type colonies (EPS(+)) have a characteristic smooth and shiny appearance which allows them to be easily distinguished from the EPS(-) variants. When grown on agar plates lacking a source of combined nitrogen, the EPS(-) variants exhibited a yellow phenotype typical of nitrogen starvation. These EPS(-) variants showed varying degrees of reversion back to the EPS(+) phenotype. After reversion, they exhibited normal diazotrophic growth on agar plates. Alcian blue and ruthenium red staining indicated that the EPS is an acidic polysaccharide, which is present as a loose network around the cell, and which can be completely removed by low speed centrifugation. The accumulation of EPS takes place mainly during the stationary phase. All EPS(-) variants failed to produce any EPS. Analysis of growth of wild-type and EPS(-) variants revealed that EPS production is beneficial for diazotrophic growth on solid medium, but not in liquid medium. In addition, EPS phenotypic alteration may have some advantage in the dispersal of cells from one place to another in the natural environment.

Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS

The organization of genes involved in utilization of methylamine (mau genes) was studied in Methylophilus methylotrophus W3A1. The strain used was a nonmucoid variant termed NS (nonslimy). The original mucoid strain was shown to be identical to the NS strains on the basis of chromosomal digest and hybridization patterns. An 8-kb PstI fragment of the chromosome from M. methylotrophus W3A1-NS encoding the mau genes was cloned and a 6,533-bp region was sequenced. Eight open reading frames were found inside the sequenced area. On the basis of a high level of sequence identity with the Mau polypeptides from Methylobacterium extorquens AM1, the eight open reading frames were identified as mauFBEDAGLM. The mau gene cluster from M. methylotrophus W3A1 is missing two genes, mauC (amicyanin) and mauJ (whose function is unknown), which have been found between mauA and mauG in all studied mau gene clusters. Mau polypeptides sequenced so far from five different bacteria show considerable identity. A mauA mutant of M. methylotrophus W3A1-NS that was constructed lost the ability to grow on all amines as sources of nitrogen but still retained the ability to grow on trimethylamine as a source of carbon. Thus, unlike M. extorquens AM1 and Methylobacillus flagellatum KT, M. methylotrophus W3A1-NS does not have an additional methylamine dehydrogenase system for amine oxidation. Using a promoter-probe vector, we identified a promoter upstream of mauF and used it to construct a potential expression vector, pAYC229.

Continuous culture studies on the synthesis of capsular polysaccharide by Klebsiella pneumoniae K1

The synthesis of capsular polysaccharide by Klebsiella pneumoniae K1 was investigated in a minimal salts medium by continuous culture. The organism produced larger amounts of polysaccharide under nitrogen-limited conditions than under carbon-limited conditions. The synthesis of polysaccharide was dependent not only on the availability of excess carbon, but also on growth rate. The rate of polysaccharide synthesis was greatest at low dilutions, low temperature (30 degrees C) and at neutral pH. Prolonged growth in nitrogen-limited culture resulted in the development of non-mucoid variants, possibly due to a selective growth advantage over mucoid cells. The non-mucoid isolate was more susceptible to some bacteriophages, possibly due to the reduction nor absence of capsular polysaccharide.

Urokinase enhances the growth of Pseudomonas spp. in vitro under nonshaking (oxygen limited) conditions.

Urokinase is a proteinase that normally functions as a plasminogen activator. It is detected in a number of tissues and can be expressed by inflammatory cells such as macrophages and polymorphonuclear leucocytes. Addition of human urokinase to cultures of mucoid or nonmucoid variants of Pseudomonas aeruginosa (strain PAO and clinical isolates from patients with cystic fibrosis) or Pseudomonas cepacia incubated in a minimal medium under nonshaking (oxygen limited) conditions led to dose-dependent enhancement of bacterial growth. The enzyme exhibited a minimal effect on the growth of bacteria when cultured under more intense aeration conditions. This enhancement of bacterial growth by urokinase required the presence of active enzyme and was not detected with inactivated enzyme or noncatalytic domains of the enzyme. Enhancement of bacterial growth was not observed following incubation of P. aeruginosa with other proteinases including thrombin, neutrophil elastase, trypsin, chymotrypsin, or pseudomonas elastase and pseudomonas alkaline proteinase. Therefore, the observed effect of urokinase was relatively specific for this enzyme. As urokinase is a natural constituent of the lung, this enzyme could contribute to bacterial growth during pulmonary infections, particularly in an inflammatory environment in which the oxygen tension may be reduced.

A Role for Exopolysaccharides in the Protection of Microorganisms from Desiccation

Mucoid strains of Escherichia coli, Acinetobacter calcoaceticus, and Erwinia stewartii were significantly more resistant to desiccation than corresponding isogenic nonmucoid mutants (survival rates of up to 35% in mucoid strains and between 0.7 and 5% in nonmucoid variants), even in colonies containing both cell types. Desiccation was found to bring about an induction of beta-galactosidase in Lon strains of E. coli K-12 carrying transcriptional lac fusions in the capsule biosynthetic (cps) regulon. This induction was dependent on the transcriptional activators RcsA and RcsB. Induction was lower in cells carrying mutations in the membrane sensor protein RcsC.

The role of saliva in aggregation and adherence of Pseudomonas aeruginosa in patients with cystic fibrosis.

The aggregation and adherence activity of P. aeruginosa, mediated by whole saliva from cystic fibrosis (CF) patients and non-CF subjects, was investigated. CF saliva-mediated aggregation of P. aeruginosa was stronger than the activity of non-CF saliva. Likewise, P. aeruginosa adherence to buccal epithelial cells (BEC) of CF patients was stronger than to BEC of non-CF subjects. Adherence of non-mucoid P. aeruginosa to BEC of CF patients was increased by saliva, whereas the mucoid variant was not. CF patients colonized with P. aeruginosa showed higher adherence of the non-mucoid variant than non-colonized CF patients. CF patients with high saliva-mediated adherence of non-mucoid P. aeruginosa also had high salivary aggregation activity. Increased CF saliva-mediated aggregation activity may be linked to the increased non-mucoid P. aeruginosa adherence to BEC of CF patients.

Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns.

Columns were packed with clean quartz sand, sterilized, and inoculated with different strains of bacteria, which multiplied within the sand at the expense of a continuous supply of fresh nutrient medium. The saturated hydraulic conductivity (HCsat) of the sand was monitored over time. Among the four bacterial strains tested, one formed a capsule, one produced slime layers, and two did not produce any detectable exopolymers. The last two strains were nonmucoid variants of the first two. Only one strain, the slime producer, had a large impact on the HCsat. The production of exopolymers had no effect on either cell multiplication within or movement through the sand columns. Therefore, the HCsat reduction observed with the slime producer was tentatively attributed to the obstruction of flow channels with slime. Compared with the results with Arthrobacter sp. strain AK19 used in a previous study, there was a 100-fold increase in detachment from the solid substratum and movement through the sand of the strains used in this study. All strains induced severe clogging when they colonized the inlet chamber of the columns. Under these conditions, the inlet end was covered by a confluent mat with an extremely low HCsat.

Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes

The susceptibility of paired mucoid and nonmucoid variants of Pseudomonas aeruginosa isolated from 13 patients with cystic fibrosis (CF) to killing by a 55,000-Da bactericidal protein (BP55) from human polymorphonuclear leukocytes was studied. Mucoid and nonmucoid variants were equally sensitive to killing by BP55 at both pH 5.6 and pH 7.2. Eleven of the isolates were resistant to the bactericidal activity of 10% normal human serum but were as sensitive as the serum-sensitive isolates to BP55. Similarly, the 15 isolates with lipopolysaccharides (LPS) containing O-polysaccharide side chains (smooth LPS) were as sensitive to BP55 as those isolates with rough LPS.P. aeruginosa isolates from patients in poor clinical condition were more likely to have LPS of the smooth type and to be resistant to killing by 10% human serum than the isolates from patients in good clinical condition. We have concluded that the susceptibility of the P. aeruginosa isolates from patients with CF to killing by BP55 does not correlate with mucoid or nonmucoid variations, with the presence or absence of smooth LPS, or with the sensitivity or resistance to killing by normal human serum.

Temperature profiles and alginate synthesis in mucoid and non-mucoid variants of Pseudomonas aeruginosa

Alginate-producing Pseudomonas aeruginosa 8821 spontaneously produced low- and high-alginate-producing variants that had low and high specific activities of GDP-mannose dehydrogenase and high and low specific growth rates, respectively. The optimal temperature for alginate production was 10°C below that for growth in the case of the high-producer but they were the same for the low-producer. In short duration thermal death experiments, alginate protected cells against lethal temperatures, although the maximal temperature for growth of the mucoid variants (43°C) was lower than that for the non-mucoid strain (44°C). The temperature profiles were associative and above 37°C growth concurred with thermal death.

Conjugal transfer of recombinant plasmids into gellan gum-producing and non-producing variants of Pseudomonas elodea ATCC 31461.

The conjugal transfer of recombinant plasmids into a gellan gum-producing, rifampicin-resistant strain of Pseudomonas elodea and into one of its non-producing variants, was studied in order to facilitate the cloning of gellan genes. The mobilization frequency of recombinant plasmids derived from pKT240, and of cosmid pJRD215 from Escherichia coli HB101 (hsdM), into the mucoid strain was below the values for the non-mucoid variant and decreased exponentially with plasmid size. Reducing the mating time on solid surfaces led to higher mobilization frequencies. Under optimal conditions, a gene bank (40-50 kb) constructed in the cosmid pJRD215 was efficiently mobilized into Gel- mutants during complementation experiments.

Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis

A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.

Whole, submandibular, and parotid saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis

The aggregation of mucoid and nonmucoid Pseudomonas aeruginosa by submandibular, parotid, and whole saliva from patients with cystic fibrosis (CF) and non-CF subjects was investigated. There were significant differences (P less than 0.01) in aggregation of mucoid and nonmucoid variants of P. aeruginosa by submandibular and whole saliva from CF patients and non-CF subjects. However, the differences in the parotid secretion were not as pronounced. Patients with CF who were colonized with P. aeruginosa demonstrated a significantly higher (P less than 0.05) percent aggregation of the mucoid variants by the submandibular secretion and of both mucoid and nonmucoid variants by whole saliva, compared with corresponding secretions from patients with CF not colonized with this pathogen. The parotid saliva aggregation activity was not markedly different for the two groups with CF. From patients with CF, whole saliva demonstrated a higher percent P. aeruginosa aggregation than did the submandibular saliva. In non-CF subjects, however, the percent aggregation of P. aeruginosa by submandibular saliva was higher than that by whole saliva. Our results indicate that the sero-mucous products of the submandibular gland have a more significant role in P. aeruginosa aggregation than the serous secreting parotid cells and that the submandibular secretion is possibly responsible for the differences in oral colonization by this pathogen in subjects with and without CF.

Comparative evaluation of mitogenicity and basement-membrane-degrading activity of Pseudomonas aeruginosa slime glycolipoprotein and alginate

Alginate from a heavily mucoid Pseudomonas aeruginosa strain and slime glycolipoprotein obtained from the revertant nonmucoid variant of the mucoid strain were tested for mitogenic activity on human peripheral lymphocytes and for degradation of 3H-labeled basement membranes of the anterior lens capsule of bovine eyes. Slime glycolipoprotein exerted mitogenic activity in concentrations from 50 to 200 micrograms/ml, whereas alginate was not mitogenic as shown by [3H]thymidine uptake. Alginate did not show any basement membrane degradation, whereas slime glycolipoprotein exhibited basement-membrane-degrading activity from 35 to 450 micrograms/ml in a dose-related manner. This activity was inhibited by metal chelators but not thiol protease inhibitors. The results suggest that alginate lacks the mitogenic and biodegrading activities of slime glycolipoprotein; these activities nevertheless need further investigation.

Active immunization of NMRI mice against Serratia marcescens. II. K-Antigen extracts

The minimal active immunogenic doses (intraperitoneal administration) of crude K-antigen extracts of Serratia marcescens for NMRI mice were 80 ng. Following ultracentrifugation (300000 × g, 16 h), supernatant fluids of three K-antigen extracts were free of contaminating DNA, RNA, and 2-keto-3-deoxy-D-manno-octonic acid (KDO), and the contents of Limulus amoebocyte lysate-reactive lipopolysaccharide (LPS) had been reduced from 100-to 1000-fold. The minimal active immunogenic doses of two ultracentrifuged K-antigen extracts were 2 and 10 μg, respectively. A mucoid strain of S. marcescens (SM 20-M; serotype O6/O14:H12) yielded nonmucoid (= SM 29-NM) variants that had lost most of the O6/O14 O-antigen (LPS) and all of the SM 29-M K-antigen extract reactivity (ELISA test) and which were ca. 5fold less mouse-virulent. Crude K-antigen extracts from S. marcescens strain SM 29-M and variant SM 29-NM failed to protect NMRI mice gainst strain SM 29-M.

Mucoid Pseudomonas aeruginosa resists nonopsonic phagocytosis by human neutrophils and macrophages.

Mucoid Pseudomonas aeruginosa is an important respiratory pathogen in patients with cystic fibrosis, and once acquired is virtually impossible to eradicate. Although mucoid P. aeruginosa is generally believed to be resistant to phagocytosis, the mechanism is not understood fully. We studied the nonopsonic phagocytosis by human neutrophils or macrophages of eight mucoid/nonmucoid P. aeruginosa pairs (three isogenic and five "wild-type"). Mucoid strains were relatively resistant to nonopsonic phagocytosis but the nonmucoid types were phagocytosis-susceptible as assessed by visual inspection and chemiluminescence assays. The mucoid and nonmucoid variants had equal numbers of pili but different surface characteristics as determined by biphasic partitioning in polyethylene glycol and dextran. The mucoid exopolysaccharide of mucoid strains appears to alter the surface characteristics of P. aeruginosa thereby rendering them resistant to nonopsonic phagocytosis. The resistance of mucoid variants of P. aeruginosa to nonopsonic phagocytosis may provide a survival advantage to these bacteria early in the course of pulmonary infection before opsonic antibody and complement are present in respiratory secretions.

Alginate production by clinical nonmucoid Pseudomonas aeruginosa strains.

The slime material from a revertant nonmucoid variant, derived by serial passage of a heavily mucoid Pseudomonas aeruginosa strain isolated from a patient with bacteremia, was found to contain 16% uronic acids, 48.5% carbohydrates, 11% protein, and 2% lipids. Chromatographic analysis by ion exchange chromatography revealed that this extracellular material consisted of three fractions, one uronic acid fraction with properties similar to those of the alginate fraction of the parental strain and two other fractions consisting of neutral sugars and proteins in approximately a 5:1 ratio. In addition, the slime material from six other clinical macroscopic nonmucoid P. aeruginosa strains was found to contain alginate. These results demonstrate that alginate production in various amounts is a property shared by all P. aeruginosa strains.

Characterization by pyocine typing and serotyping of oral and sputum strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients.

Oral and sputum isolates of Pseudomonas aeruginosa in patients with cystic fibrosis were investigated. Of the 17 patients studied, 12 patients (71%) yielded both mucoid and nonmucoid variants of Pseudomonas aeruginosa from sputum and (or) various oral ecological sites, such as buccal mucosa, tongue dorsum, dental plaques, and saliva. A total of 51 strains of mucoid and nonmucoid Pseudomonas aeruginosa were isolated from these patients and were phenotypically characterized by both pyocine typing and serotyping. Five patients (42%) were colonized or infected by a single strain of Pseudomonas aeruginosa, whereas 7 patients (58%) were cocolonized or coinfected by two or more phenotypically different strains of Pseudomonas aeruginosa. To understand the mechanisms involved in Pseudomonas aeruginosa colonization, it may be necessary to identify multiple isolates of Pseudomonas aeruginosa not only from the sputum but also from the various oral ecological sites and to further explore the role of the oral cavity in this colonization.

Interbacterial adhesion between Pseudomonas aeruginosa and indigenous oral bacteria isolated from patients with cystic fibrosis.

Interbacterial adhesion between strains of Pseudomonas aeruginosa and strains of indigenous oral bacteria, both of which were isolated from the oral cavity of cystic fibrosis patients, was investigated by the phenomenon of the coaggregation reaction. A total of 22 strains of P. aeruginosa were isolated from the oral cavity of 17 patients and examined for their abilities to coaggregate with 5 strains each of Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii. Coaggregation reactions were common between these oral bacteria and both the mucoid and nonmucoid variants of P. aeruginosa. All strains of P. aeruginosa were also able to agglutinate neuraminidase-treated or untreated human erythrocytes of blood types A, B, and O. Positive coaggregation reactions were further characterized by determining the effects of several sugars, and of heat and protease treatments of the bacteria. None of the coaggregtion reactions were inhibited by 0.05 M lactose, galactose, glucose, fucose, or mannose. All coaggregation reactions were dependent upon heat- and protease-sensitive components of the Pseudomonas. Thus, the interbacterial adhesions between P. aeruginosa and the oral bacteria studied appears to involve adhesins on the Pseudomonas cell, which bind to complementary receptors, on the cell surfaces of oral bacteria. The apparent prevalence and diversity of interbacterial adhesions between P. aeruginosa strains originating from the oral cavity of cystic fibrosis patients and strains of the indigenous oral bacteria suggest that some of these reactions may affect the extent to which P. aeruginosa colonizes in the oral cavity of cystic fibrosis patients, and thereby, influence susceptibility of the host to infection.

Role of pili in the adherence of Pseudomonas aeruginosa to injured tracheal epithelium.

Pili have been demonstrated to be the adhesins of nonmucoid Pseudomonas aeruginosa for buccal cells. In this study, we examined their role in the adherence of both mucoid and nonmucoid strains to injured tracheal cells. Pili incubated with tracheal cells inhibited the adherence of a nonmucoid strain in a dose-dependent manner. Both homologous and heterologous pili inhibited this nonmucoid strain. Antibody against pili from the nonmucoid strain inhibited adherence of the homologous but not a heterologous strain. Pili failed to inhibit two mucoid strains, but inhibited nonmucoid variants derived from mucoid strains. These studies suggest that pili mediate the adherence of nonmucoid strains to injured tracheal cells but that they are not the final mediators of adherence of mucoid strains. It is also inferred that there are differences in the receptor for mucoid and nonmucoid strains.

Protease production by Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The temporal appearance of extracellular proteases produced by Pseudomonas aeruginosa was analyzed by pH 9 and pH 4 polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Ammonium sulfate precipitates of culture supernatants from various stages of growth revealed a time-dependent increase in number and amount of proteolytically active proteins. One mucoid P. aeruginosa clinical isolate and its derived nonmucoid variant, as well as two other nonmucoid variant P. aeruginosa strains (all from cystic fibrosis patients), showed similar production of five differently migrating proteases (P1 to P5, numbered according to increasing net negative charge) in pH 9 PAGE and one protease in pH 4 PAGE. P2, P3, and P5 increased to maximum concentrations at 24 to 48 h, decreasing thereafter, whereas P4 continued increasing even at 83 h, and P1 fluctuated. P3 was identified as an elastase. P2 was possibly composed of polypeptide chains bridged by disulfide bonds, since without reduction it migrated in sodium dodecyl sulfate-PAGE as a single protein, and with reduction it migrated as three protein bands. Two-dimensional PAGE revealed multiple molecular weight species within protease-positive bands in pH 9 gel strips. Isoelectric focusing gave a pattern of protein separation that correlated with two-dimensional PAGE analysis. Thus, greater heterogeneity of active proteases than previously reported has been demonstrated in all P. aeruginosa clinical isolates studied by sensitive two-dimensional PAGE analysis.