Structural studies of acidic glucomannans from strains of Serratia marcescens O14 and O6

Partially acetylated acidic glucomannans have been isolated from three strains of Serratia marcescens serogroup O14 and one strain of the cross-reacting serogroup O6. Degradative and spectroscopic studies established that the polysaccharides have branched tetrasaccharide repeating-units of the structure shown. Individual polymers may vary in the extent or location of O-acetylation, and in the extent of undefined heterogeneity apparently associated with the glucosyluronic acid residues. Although the polymers were obtained from lipopolysaccharide extracts, there are indications of a (micro)capsular origin. The acidic glucomannans may constitute a common antigen which defines the O14-O6 complex of S. marcescens.     

Isolation and characterization ofSerratia marcescens mutants defective in prodigiosin biosynthesis

Mutants deficient in the biosynthesis of prodigiosin have been obtained by treatingSerratia marcescens with high doses of ultraviolet radiation. Mutants were selected on the basis of the color characteristics of their colonies when grown on peptone glycerol medium. New types of mutants, with unusual blocks in the biosynthetic pathway of prodigiosin, were obtained. All the mutants were classified under a new scheme on the basis of the syntrophic pigmentation characteristic and infrared spectroscopic analysis of their pigment. By these criteria mutants could be distinguished into eight distinct classes. Classes I to III include mutants of the three classes (M1, B3, and B1) reported previously [Morrison, DA (1966) J Bacteriol 91:1599–1604] and several new ones. Mutants blocked in the methylamylpyrrole (MAP) arm of the bifurcated pathway were assigned to class I. A class II mutant was distinguished by its inability to synthesize methoxybipyrrolecarboxyaldehyde (MBC), but was able to produce norprodigiosin. Class III mutants were deficient in the synthesis of hydroxybipyrrolecarboxaldehyde (HBC). Double mutants were obtained with defects in the expression of both MBC and MAP and were assigned to class IV. Mutants of class V were unable to synthesize HBC and MAP, but could form MBC when furnished with exogenous HBC. Class VI and VII mutants were defective in the synthesis of all three precursors, but differed in their ability to perform the coupling step. Finally, a mutant of class VIII was found to produce the three intermediates, but was deficient in prodigiosin or norprodigiosin biosynthesis, indicative of a defect in the enzymatic condensation of MAP with the bipyrroles MBC and HBC. The anomalous pattern of syntrophism among certain interclass mutants suggests that the physiology of pigment formation inS. marcescens is quite complex.     

Transport of basic amino acids and regulation of aspartate kinase activity in thialysine andL-canavanine resistant strains ofSerratia marcescens

Growth ofSerratia marcescens was not inhibited by high concentrations ofL-lysine and its structural analogues,L-canavanine and S-(2-aminoethyl)-L-cysteine (thialysine). This insensitivity was not caused by deficient transport of basic amino acids, unlike in mutant strains ofEscherichia coli having the same properties. The tested strains showed a lack of regulation at the aspartate kinase level towardL-lysine and thialysine. The data indicate great intraspecific variability for aspartate kinase regulation inS. marcescens.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Structural studies of glucorhamnans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O4 and O7, and of an O14 strain

Partially acetylated glucorhamnans have been isolated from the lipopolysaccharides of three strains of Serratia marcescens. The polymer from the reference strain (C.D.C. 864-57) for serogroup O4 has the disaccharide repeating-unit shown below, in which acetylation at position 2 of the rhamnosyl residue is approximately 90% complete. Similar glucorhamnans from the reference strain (C.D.C. 843-57) for serogroup O7 and from a pigmented strain (NM) of serogroup O14 differ only in the configuration of the L-rhamnopyranosyl residue (beta) and the extent of O-acetylation (O7, almost stoichiometric; NM, 80-90%). Glucorhamnans of the second type have been isolated previously from the lipopolysaccharides of other strains of S. marcescens, including the reference strain for serogroup O6 and another pigmented O14 strain (N.C.T.C. 1377). In all cases, the lipopolysaccharide extracts also contained acidic glycans, but the glucorhamnans are believed to constitute the integral side-chains. (Formula: see text).     

Hemagglutination (fimbriae) and hydrophobicity in adherence ofSerratia marcescens to urinary tract epithelium and contact lenses

The capacity of 59 isolates of Serratia marcescens, obtained from urinary tract infections, wounds, and contact lenses or their paraphernalia, to agglutinate erythrocytes from different animal species was tested. Three main patterns were found: mannose-sensitive agglutination of guinea-pig, fowl or horse erythrocyte; mannose-resistant agglutination of chicken or pigeon erythrocytes alone or in combination with mannose-sensitive agglutination; and no agglutination. Hemagglutination capacity was associated with isolates from urinary tract infection, but not with isolates associated with contact lenses. Adherence to human urinary tract epithelium did not correlate with the hemagglutination patterns nor with the origin of the isolates. Some strains of different hemagglutination pattern were selected for the study of hydrophobicity and adherence to contact lens polymers. Hydrophobicity, as determined by degree of partition in hexadecane and water (BATH-values), correlated neither with degree of adherence to contact lens polymers nor with the hemagglutination pattern. For a representative strain there was an excellent correlation (r2 = 0.98) between adherence and the water content (hydrophobicity) of the lens polymers. These results suggest that, as with tissues, other factors interact with hydrophobicity in causing adherence to plastics.     

Structure of a neutral glycan from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O2 and O3

Serogroups O2 and O3 of Serratia marcescens are differentiated by acidic glycans present in the aqueous phase when lipopolysaccharides are extracted from the reference strains by the aqueous-phenol method. The phenolic phases of these extracts from both strains also contain lipopolysaccharides, from which the same neutral glycan is released on milk acid hydrolysis. The neutral glycan has the disaccharide repeating-unit shown, and accounts for the cross-reactions between the two serogroups and also with serogroup O21: --> 4)-alpha-D-GlcpNAc-(1-->4)-beta-D-ManpNAc-(1--.     

Molecular heterogeneity of heat-stable toxin-producing and non-producing Escherichia coli O25 strains isolated in Japan

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.     

Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep.

Lipopolysaccharide (LPS) from 40 isolates of Pasteurella haemolytica, comprising 23 serotype A1, seven serotype A2, one serotype T4, one serotype T10 and eight untypable isolates, obtained from diseased and healthy cattle or sheep, was characterized by SDS-PAGE and Western blotting. Ten different SDS-PAGE LPS profiles, five smooth and five rough, were identified among the biotype A and untypable isolates and designated LPS types 1-10. LPS types 1 and 2 were smooth, had similar O-antigen banding-patterns but differed in the low-molecular-mass or core-oligosaccharide regions; type 3 LPS was rough but had a core-oligosaccharide region similar to that of LPS type 1. No similarities were observed between these LPS types and types 6, 7 and 9, which were smooth, and types 4, 5, 8 and 10, which were rough. Most serotype A1 isolates (19/23) were of LPS type 1, whereas two isolates each had LPS of types 2 and 3. The majority (5/7) of serotype A2 isolates possessed type 3 LPS, whereas the remaining two isolates each had LPS of types 4 and 5. There was much greater heterogeneity within the untypable group of isolates, which comprised LPS of types 1 and 9 (two isolates each), and 6, 7, 8 or 10 (one isolate each). Western blotting analysis demonstrated that LPS types 1 and 2 had immunologically identical O-antigen side-chains but differed in their core-oligosaccharide regions, whereas the core-oligosaccharide region of rough LPS type 3 was immunologically very similar to that of LPS type 1. The other LPS types were immunologically unrelated to these three LPS types. The majority (20/23) of serotype A1 isolates originated from cattle and possessed LPS types 1 or 2, different from most (5/7) of the serotype A2 isolates which originated from sheep and possessed LPS of types 3 or 4. However, two of the three ovine serotype A1 isolates had the same type 3 LPS as occurred in most of the ovine serotype A2 isolates, suggesting a possible correlation between LPS type and host specificity. This study has demonstrated that LPS diversity within different serotypes of P. haemolytica is greater than was previously thought and that certain LPS types might be host-specific.     

Intra-strain heterogeneity in expression of lipopolysaccharide by strains of Salmonella virchow

Strains of Salmonella virchow express long-chain lipopolysaccharide which migrates as one of two distinct profiles during SDS-PAGE. Both LPS phenotypes were detected within a given strain of Salm. virchow and shared the O = 7 antigen.     

Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10(4.61) to 10(7.11)), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.     

Enhancement by Sodium Dodecyl Sulfate of Pigment Formation in Serratia marcescens O8

Three methods were used to determine the enhancement by sodium dodecyl sulfate (SDS) of prodigiosin formation in Serratia marcescens O8. The results of the agar disk diffusion method indicated that pigment formation was dependent upon the concentration of SDS. Diameters of the pigment zones were proportional to the logarithm of SDS concentrations of 300 to 1,500 μg/ml. When bacteria were grown in broth containing SDS from 0 to 800 μg/ml and the pigment extracts were analyzed spectrophotometrically, a similar enhancement of pigment formation was observed. Finally, these results were confirmed by high-performance liquid chromatographic analysis of the extracts. Prodigiosin appeared to be the sole component with increased synthesis. The possible mechanism of the SDS enhancement effect could be explained by an increase in negative binding sites by the association of SDS with a cell envelope component(s). These binding sites may be required for prodigiosin synthesis.     

New R-plasmids in strains of Serratia marcescens with multiple drug resistance

In 4 S. marcescens polyresistant strains isolated from patients conjugative plasmids transferred to Escherichia coli have been detected. Two of these strains carry each one plasmid which codes resistance to 10 different antibiotics, including aminoglycosides which rarely occur in our country, and belongs to group IncC. The third strain is the host of 2 plasmids. One of them is similar to the above-mentioned 2 plasmids with respect to the incompatibility group and a set of markers, but additionally codes resistance to cephalosporins; the second plasmid has been determined as belonging to group IncM, unstable and capable of rendering the cells highly resistant only to aminoglycosides. And, finally, the fourth strain also carries 2 plasmids: one of them is unstable and belongs, supposedly, to group IncI alpha, and the second plasmid is stable and belongs to group IncM. The plasmid of group IncI alpha differs from all other plasmids of our Serratia by its capacity of rendering the cells highly resistant to chloramphenicol.     

Genomic comparison of plant pathogenic and nonpathogenic Serratia marcescens strains by suppressive subtractive hybridization

Cucurbit yellow vine disease (CYVD) is caused by disease-associated Serratia marcescens strains that have phenotypes significantly different from those of nonphytopathogenic strains. To identify the genetic differences responsible for pathogenicity-related phenotypes, we used a suppressive subtractive hybridization (SSH) strategy. S. marcescens strain Z01-A, isolated from CYVD-affected zucchini, was used as the tester, whereas rice endophytic S. marcescens strain R02-A (IRBG 502) was used as the driver. SSH revealed 48 sequences, ranging from 200 to 700 bp, that were present in Z01-A but absent in R02-A. Sequence analysis showed that a large proportion of these sequences resembled genes involved in synthesis of surface structures. By construction of a fosmid library, followed by colony hybridization, selection, and DNA sequencing, a phage gene cluster and a genome island containing a fimbrial-gene cluster were identified. Arrayed dot hybridization showed that the conservation of subtracted sequences among CYVD pathogenic and nonpathogenic S. marcescens strains varied. Thirty-four sequences were present only in pathogenic strains. Primers were designed based on one Z01-A-specific sequence, A79, and used in a multiplex PCR to discriminate between S. marcescens strains causing CYVD and those from other ecological niches.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Properties of Serratia marcescens strains isolated in septic diseases of newborn infants

As the result of the study of blood and liquor samples from 120 newborns, Serratia marcescens was isolated in 21 cases (17.5 %). 8 strains were isolated from the environment of these patients. Almost all strains isolated from both the patients and the environment (with the exception of one environmental strain) belonged to serotype 04. The isolated S. marcescens strains were resistant to penicillin, ampicillin, streptomycin, kanamycin, oxacillin, methicillin, ceporin and moderately sensitive to polymixin. 2 strains from the environment and 9 strains from the patients were mildly sensitive to gentamicin. In one hospital all isolated strains were found to have 2 transmissive R plasmids with the molecular weight 40 and 60 megadaltons. The presence of R plasmids with the same molecular weight in all S. marcescens strains isolated in this hospital, as well as their serological identity, suggest that in all patients infection originated from a common source.