Non-pigmented Strains Research Articles

Culture-based methods for detection and identification of Streptococcus agalactiae in pregnant women—what are we missing?

We aimed to compare BD group B Streptococcus differential agar (GBSDA) with inoculation into LIM broth and subculture onto blood agar plates (LIM-BA) as a method for GBS screening in pregnant women. First, we compared the detection threshold and the ease of use of GBSDA with LIM-BA by inoculating known numbers of GBS mixed with approximately 10(5) cfu of other bacteria. Second, we tested the production of carotenoid pigment in 155 GBS blood culture isolates. Finally, we compared GBSDA, LIM broth with direct GBS antigen detection (LIM-AG) and LIM-BA as methods for GBS screening in pregnant women. GBS colonies were easily detected on GBSDA at a threshold of 20 cfu. In contrast, GBS was not detected in a mixed culture from LIM broth with initial inocula of up to 100 cfu. Orange pigment was produced in 146/155 GBS blood culture isolates. Pigment was not produced in eight non-hemolytic and two hemolytic strains. GBS was detected by GBSDA in 58 out of 297 parturient women (19.5%) but in only 50 (16.8%, P > 0.05) and 46 (15.4%, P = 0.079) women by LIM-BA and LIM-AG, respectively. GBSDA is a satisfactory medium for the rapid detection of most GBS isolates, but other methods may be needed if detection of all non-pigmented strains is required.

Burkholderia cenocepacia C5424 Produces a Pigment with Antioxidant Properties Using a Homogentisate Intermediate

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H2O2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Heat and pulsed electric field resistance of pigmented and non-pigmented enterotoxigenic strains of Staphylococcus aureus in exponential and stationary phase of growth

The survival of four enterotoxigenic strains of Staphylococcus aureus (with different pigment content) to heat and to pulsed electric fields (PEF) treatments, and the increase in resistance to both processing stresses associated with entrance into stationary phase was examined. Survival curves to heat (58 °C) and to PEF (26 kV/cm) of cells in the stationary and in the exponential phase of growth were obtained. Whereas a wide variation in resistance to heat treatments was detected amongst the four strains, with decimal reduction time values at 58 °C ( D 58 °C) ranging from 0.93 to 0.20 min, the resistance to PEF was very similar. The occurrence of a higher tolerance to heat in stationary phase was coincident with a higher content in carotenoid pigmentation in S. aureus colonies. However, cells of the most heat resistant (pigmented) and the most heat sensitive (non-pigmented) strains in the mid-exponential phase of growth showed similar resistance to heat and to PEF. Therefore the increase in thermotolerance upon entrance into stationary phase of growth was more marked for the pigmented strains. Recovery in anaerobic conditions particularly enhanced survival to heat treatments in a non-pigmented strain. Strain CECT 4630, which possess a deficient sigma B activity, showed low heat resistance, low pigmentation, and reduced increase in thermotolerance in stationary phase. These results indicate that the magnitude of the development of a higher heat resistance in S. aureus in stationary phase is positively related to the carotenoid content of the strain. The development of tolerance to pulsed electric field was less relevant and not linked to the carotenoid content.

Propionibacterium jensenii Produces the Polyene Pigment Granadaene and Has Hemolytic Properties Similar to Those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.

Evaluación de la capacidad invasiva de las micobacterias no pigmentadas de crecimiento rápido mediante el estudio de la morfología de las microcolonias en fibroblastos

The invasive capacity of rapidly-growing nonpigmented mycobacteria strains was evaluated by means of a fibroblast microcolony assay and related to the colony phenotype on Middlebrook 7H11 and to the clinical significance of the isolates. Twenty-nine strains [Mycobacterium chelonae (8), M. fortuitum (6), M. peregrinum (5), M. abscessus (5), M. mucogenicum (4) and M. septicum (1)], proceeding from a bacterial collection and clinical isolates, were evaluated. The smooth or rough phenotype of the colonies was assessed in Middlebrook 7H11 medium. Intracellular invasiveness was determined by the fibroblast-microcolony assay described by Shepard. Quantitative culture characteristics were compared with Student's t-test, and qualitative characteristics with Fisher's exact test. No significant differences were found between colonies with different phenotypes or strains having a different clinical significance, except for two strains of Mycobacterium chelonae isolated from cases of catheter-related bacteremia, which showed elongated microcolonies. M. fortuitum and M. peregrinum strains showed larger microcolonies than M. chelonae, M. abscessus and M. mucogenicum, and displayed a fluffy appearance, while the latter two strains showed rounded colonies. Very few strains of mycobacteria had invasive capacity and the majority of strains isolated from human infections do not show this characteristic; hence this trait is not essential for mycobacteria to cause infection in humans.

Choline acetyltransferase immunoreactive cortical interneurons do not occur in all rodents: A study of the phylogenetic occurrence of this neural characteristic

The present study was designed to provide results aimed at testing whether the interneurons with choline acetyltransferase immunoreactivity (ChAT), probably representing GABA interneurons, found in the cerebral cortex of the rat represent a common feature of the order Rodentia. Initially we verified the presence of ChAT immunoreactive bipolar cell bodies, axons and terminal-like fibres in pigmented (Long-Evans) and non-pigmented (Sprague-Dawley) strains of Rattus norvegicus, confirming that the ChAT polyclonal antibodies (AB144P and AB143, Chemicon; VChAT, Sigma) with the immunohistochemical techniques used provided the same staining as previously described for this species. We then examined pigmented (AKR3) and non-pigmented (C3H) strains of Mus musculus, wild caught striped mice ( Rhabdomys pumilio), bushveld gerbil ( Tatera brantsii), greater canerat ( Thryonomys swinderianus) and common molerat ( Cryptomys hottentotus). The AB144P antibody revealed cortical interneurons in both strains of M. musculus and in R. pumilio, but not in the other species. In all species/strains cortical ChAT immunoreactive axons and terminal-like fibres were localized with the AB144P antibody. In the non- Rattus species/strains there was no evidence for localization of ChAT immunoreactivity in any cortical cell bodies using the AB143 and vesicular acetylcholine transporter (VChAT) antibodies despite extensive localization in axons and terminal-like fibres. It is concluded that bipolar cortical GABA interneurons in certain rodent species may develop ChAT immunoreactivity but not VChAT immunoreactivity making the cholinergic relevance of ChAT in the GABA interneurons uncertain and may exclude these neurons from being part of the traditionally defined cholinergic system.

Metabolic and regulatory engineering of Serratia marcescens: mimicking phage-mediated horizontal acquisition of antibiotic biosynthesis and quorum-sensing capacities

Serratia marcescens is an important cause of opportunistic human infections. Many, but not all, strains produce prodigiosin, a secondary metabolic, red-pigment antibiotic, the biosynthesis of which is directed by the pig gene cluster. Quorum sensing (QS) involves the production and detection of chemical signal molecules as a means to regulate gene expression in response to population cell density. Several strains of S. marcescens have previously been shown to possess an N-acyl-L-homoserine lactone (aHSL) QS system. This study aimed to determine the impact of introducing, by phage-mediated horizontal gene transfer, a biosynthetic gene cluster (pig) and a regulatory locus (aHSL QS) into strains lacking the respective trait. The pig cluster from S. marcescens ATCC 274 (Sma 274) was transferred to the non-pigmented strain, S. marcescens strain 12 (Sma 12). In the engineered strain, pigment was expressed and brought under the control of the recipient's native regulatory systems (aHSL QS and luxS). Moreover, transfer of the aHSL locus from Sma 12 to the non-QS Sma 274 resulted in the imposition of aHSL control onto a variety of native traits, including pigment production. In addition, during this study, the QS regulon of the clinical strain, Sma 12, was characterized, and some novel QS-regulated traits in S. marcescens were identified. The results have implications for the evolution and dissemination of biosynthetic and QS loci, illustrating the genetic modularity and ease of acquisition of these traits and the capacity of phages to act as vectors for horizontal gene transfer.

Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule-forming and nitrogen-fixing bacteria

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C(1) compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060(T)=CNCM I 2342(T)=LMG 21967(T)) is proposed for this group of root-nodule-forming bacteria.

Optimization of medium and cultivation conditions for pullulan production by a new pullulan-producing yeast strain

Yeast strain Y68, producing a large amount of pullulan, was isolated from the leaves collected in the south of China. This strain was identified to be Rhodotorula bacarum by BIOLOG analysis and routine yeast identification. This is the first time to report that pullulan was produced by R. bacarum. The optimal medium for pullulan production by this strain was 8.0% (w/v) glucose, 2.0% (w/v) soybean cake hydrolysate, 0.5% (w/v) K 2HPO 4, 0.1% (w/v) NaCl, 0.02% (w/v) MgSO 4·7H 2O, 0.06% (w/v) (NH 4) 2SO 4, pH 7.0. The optimal cultivation conditions for pullulan production by this strain in 300-ml shake flask containing 50 ml of medium were observed at 28 °C and with 180 rpm. Under these conditions, 5.9% (w/v) pullulan was produced within 60 h. This was the highest pullulan yield produced by yeasts obtained so far. No pigment in the medium was observed during the fermentation, suggesting that strain Y68 was a non-pigmented yeast strain.

Pseudoepidemic due to a unique strain of Mycobacterium szulgai: genotypic, phenotypic, and epidemiological analysis.

In mid-1999, we noted multiple isolations at the Veterans Affairs Medical Center (VAMC) Houston Tex. of an unusual nonpigmented Mycobacterium species. Since, on the basis of 16S rRNA gene sequence analysis, the strains were identical to the Mycobacterium szulgai type strain and since M. szulgai has been reported only rarely as a commensal or environmental isolate, we were concerned about laboratory contamination, nosocomial spread, or even the possibility that this could be a novel organism associated with disease. Our investigation found that from 1999 to 2000, 37 strains of M. szulgai were isolated from patients at the VAMC (the base rate for the previous 10 years had been <1 isolation per year). We compared the phenotypic properties and genetic relatedness of these 37 strains (31 of which were nonpigmented) as well as eight stock strains and the M. szulgai type strain. All strains were similar in cellular fatty acid patterns, growth rates, and biochemical characteristics. However, we found three genogroups by gene sequence analysis. Genogroup I comprised the M. szulgai type strain, all the tested nonpigmented strains (27 of the 31 strains were tested), two pigmented strains isolated in 1999 and 2000, and five pigmented stock strains. Genogroup II comprised five pigmented strains: three were isolated from 1999 to 2000 and two were stock strains. The single strain (isolated in 1996) in genogroup III was pigmented and was the only strain associated with disease. Whereas the randomly amplified polymorphic DNA (RAPD) patterns of all nonpigmented strains were identical, indicating that they came from a common source (the pseudoepidemic strain), the RAPD patterns of the other strains were varied. In our investigation for a possible source, we found that there were no common reagents, specimen-processing or patient locations, or procedures linking the 31 pseudoepidemic strains. However, a nonpigmented M. szulgai strain with a gene sequence and RAPD pattern identical to those of the pseudoepidemic strain was recovered from a water storage tank serving the hospital. We concluded that the strains most likely originated from hospital water, which transiently inoculated our patients. Although no disease was associated with this cluster of isolates, the event was costly because identification was problematic and we could not easily discount the isolations, since most of the patients were immunocompromised and were candidates for opportunistic infection.

Plants in the pink: cytokinin production by methylobacterium.

Bacteria of the genus Methylobacterium are well-studied examples of facultative methylotrophs. These bacteria are classified as α-proteobacteria and are capable of growth on methanol and methylamine as well as on a variety of C2, C3, and C4 compounds (8). Methylobacterium strains are commonly found in soils, as well as on the surfaces of leaves of a wide variety of plants (1). Because of their distinctive pink pigmentation, they are sometimes referred to as PPFMs (pink-pigmented facultative methylotrophs). Evidence exists that these bacteria utilize methanol emitted by the stomata of plants (9), but the details of their relationship to plants has been unclear. Are Methylobacterium strains commensal bacteria, or do they communicate with plants in a more intimate relationship? A number of reports suggest that Methylobacterium strains are more than passive passengers on plant leaves. For instance, they have been shown in some cases to stimulate seed germination and plant development, possibly by production of phytohormones (2, 3, 4), and it has been reported that one strain produces the cytokinin zeatin (5) while others have been reported to produce indole acetic acid (6). In addition, Methylobacterium strains have been suggested to contribute to the flavor of strawberries (12) and have been localized as endosymbionts within cells of the buds of Scotch pine (Pinus sylvestris) (10). One nonpigmented Methylobacterium strain has been shown to form a root-nodulating nitrogen-fixing symbiosis with a legume (11). A partial genome sequence of Methylobacterium extorquens AM1 is available (http://pedant.mips.biochem.mpg.de/), which reveals a number of open reading frames with significant identity to genes involved in plant association in rhizobia and Agrobacterium (M. Lidstrom, unpublished results). Therefore, at this point numerous indications exist that these PPFMs interact with plants, but the biochemical and genetic details of those interactions have remained elusive. In this issue, Koenig et al. (7) present a set of studies that represents an important step in understanding the molecular basis of Methylobacterium-plant interactions. They show that four different Methylobacterium strains representing leaf isolates and a type M. extorquens strain all produce the cytokinin trans-zeatin at low levels in pure culture and excrete it into the culture medium. Biochemical and genetic evidence is presented suggesting that the trans-zeatin is not synthesized de novo but instead is derived from tRNA. This result is surprising, since so far the tRNA-derived zeatin from bacteria and plants has been restricted to the cis isomer. Is the low-level production of trans-zeatin observed in these strains sufficient to have an effect on plants? Surprisingly, the Methylobacterium-specific stimulation of seed germination that has been described in the past (3, 4) was unaffected in a mutant that is incapable of producing trans-zeatin. Thus, the answer to this question is still outstanding, as is the source of the stimulation effect itself. However, the results presented by Koenig et al. provide mechanistic evidence for cytokinin production by Methylobacterium strains, leading the way for future studies of the Methylobacterium-plant relationship. In addition, this evidence that a commensal bacterium produces a phytohormone via a previously unknown route provides new insights into the role of commensal plant bacteria in general.

Identification and characterization of a putative transcriptional regulator controlling the expression of fouling inhibitors in Pseudoalteromonas tunicata.

The dark green pigmented marine bacterium Pseudoalteromonas tunicata colonizes living surfaces and produces a range of extracellular compounds that inhibit common fouling organisms, including marine invertebrate larvae, algae, bacteria, and fungi. We have observed a positive correlation between the antifouling activity of P. tunicata strain D2 and the expression of pigmentation. To address the hypothesis that pigmentation and antifouling may be jointly regulated in this organism and to begin to identify potential regulatory elements, we used transposon mutagenesis to generate a strain of P. tunicata deficient in antifouling activity. The data presented here describe the phenotypic and molecular characterization of a nonpigmented transposon mutant strain of P. tunicata (D2W2). Analyses of the antifouling capabilities of D2W2 demonstrate that this strain is deficient in the ability to inhibit each of the target fouling organisms. Genetic analysis of D2W2 identified a gene, designated wmpR (white mutant phenotype), with high sequence similarity to transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Two-dimensional polyacrylamide gel electrophoresis analysis revealed that WmpR is essential for the expression of a significant subset of stationary-phase-induced proteins likely to be important for the synthesis of fouling inhibitors. The identification of a gene involved in the regulation of expression of antifouling phenotypes will contribute to the understanding of the interactions between bacteria and other surface-colonizing organisms in the marine environment.

Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens.

Eleven non-pigmented strains of gram-negative, aerobic, marine bacteria with polar flagella were isolated from the thallus of the brown alga Fucus evanescens collected in the Kraternaya Bight of the Kurile Islands in the Pacific Ocean. These organisms were conspecific and exhibited high levels of genetic relatedness (up to 91%). The G+C contents of the DNAs of these strains were 42.9-43.3 mol%. These halophilic bacteria had bacteriolytic, proteolytic and haemolytic activities and degraded algal polysaccharides, synthesizing a number of glycoside hydrolases (fucoidanases, laminaranases, alginases, agarases, pullulanases, beta-glucosidases, beta-galactosidases, beta-N-acetylglucosaminidases and beta-xylosidases). By 16S rDNA analysis, the bacteria were shown to belong to the genus Pseudoalteromonas, a member of the gamma-subclass of the Proteobacteria. DNA from the strains isolated from the brown alga showed 27-54% genetic relatedness with respect to DNAs of other type strains of the genus Pseudoalteromonas. The phenotypic characteristics, together with the genetic evidence, indicate that this group of epiphytic bacteria represents a distinct species, Pseudoalteromonas issachenkonii sp. nov., for which the type strain is KMM 3549T (= LMG 19697T = CIP 106858T).

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture.

The production of pullulan from beet molasses by a pigment-free strain of Aztreobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by 13C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (Mw = 2.1-4.1 x 10(5)), root mean square of the radius of gyration (R = 30-38 nm), and polydispersity index (Mw/Mn = 1.4-2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8-10.0 (w/v), the solution's rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s(-1) at 20 degrees C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.

Molecular identification of a clinical isolate of non-pigmented and asporogenic Aspergillus fumigatus strain which cannot be identified by traditional morphological methods.

A non-pigmented and asporogenic fungal strain without taxonomically useful characteristics was isolated from sputum of a patient with chronic bronchitis. Although the taxonomic position could not be determined based on traditional morphological criteria, the fungus was identified as an Aspergillus fumigatus mutant strain by molecular techniques including ITS sequence analysis and PCR identification system using a PCR primer pair specific for A. fumigatus. Usefulness of these molecular techniques for the identification of non-classifiable fungal strains in a clinical laboratory is discussed.

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov. — Novel Aerobic Facultatively Methylotrophic Bacteria Utilizing Dichloromethane

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).

Clinical relevance and virulence factors of pigmented Serratia marcescens.

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD(50)=10(7) bacteria ml(-1)) and showed virulence factors, but were isolated with low frequency (3. 4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.

Clinical relevance and virulence factors of pigmented Serratia marcescens

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD 50=10 7 bacteria ml −1) and showed virulence factors, but were isolated with low frequency (3.4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.

Response of Escherichia coli Containing Mycobacterial Carotene Genes to UV Radiation.

The plasmid pC5, which encodes biogenesis of lycopene in Mycobacterium aurum A+, was partially digested by restriction endonucleases and generated fragments were cloned. After transformation of Escherichia coli (colorless bacteria) with the plasmids so constructed, seven orange clones were detected and found to carry the same recombinant plasmid (pC51). E. coli cells containing this plasmid synthesize neurosporene and lycopene, and were more resistant to ultraviolet irradiation than nonpigmented strain.

Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture

The production of pigment-free pullulan by Aureobasidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l −1), polysaccharide productivity (4.5 g l −1 per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l −1) and pullulan productivity (3.5 g l −1 per day) were obtained in culture grown with feeding substrate containing 50 g l −1 sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly α-(1→4) (∼66%) and α-(1→6) (∼31%) glucosidic linkages; however, a small amount (<3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by 13C-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.