Transduction of fimbriation demonstrating common ancestry in FIRN strains of Salmonella typhimurium.

The production of fimbriate (Fim+) recombinants was observed in transductional crosses between different pairs of wild-type strains of different biotypes of Salmonella typhimurium. Fim+ recombinants were readily produced in transductions from Fim+ donor strains to Fim- recipient strains and, less frequently, between some pairs of Fim- strains, for example, between almost any strain of the firn biogroup (Fim- Inl- Rha- Bxyl-) and many strains of the non-FIRN Fim- biogroup. None of numerous crosses between different pairs of FIRN strains gave Fim+ recombinants, suggesting that the fim mutation was present at the same intragenic site in all FIRN strains. FIRN strains are thought to have descended from a single ancestral FIRN bacterium which originated by a series of mutations from a strain of the common biotype 1a (Fim+ Inl+ Rha+ Bxyl+). Two FIRN-like (Fim- Inl+ Rha- Bxyl-) strains that did not yield Fim+ recombinants in crosses with FIRN strains were probably wild-type Inl+ mutants from FIRN strains.     

Selective outgrowth of fimbriate bacteria in static liquid medium

Competitive mixed cultures were grown from inocula of a large number of bacteria of a genotypically nonfimbriate (fim(-)) strain of Salmonella typhimurium and a small number of a genotypically fimbriate (fim(+)) variant strain that formed type 1 fimbriae and had been derived from the fim(-) strain by phage transduction. The fim(+) strain differed from the fim(-) strain in fermenting l-rhamnose (rha(+)), and the viable fim(+) and fim(-) bacteria present in the cultures after different periods at 37 C were counted differentially in platings on rhamnose media. When the cultures were grown under aerobic static conditions in tubes of nutrient broth, the fim(+) bacteria rapidly outgrew the fim(-) bacteria, so that, although starting as a small minority (e.g., 1 in 10(7)), they approached or surpassed the number of the fim(-) in 48 hr. A pellicle consisting of fimbriate bacteria was formed on the surface of the broth between 6 and 24 hr, and it is thought that the advantage of access to atmospheric oxygen enjoyed by these bacteria in the pellicle enabled them to outgrow the fim(-) bacteria confined in the oxygen-depleted broth. The fim(+) bacteria did not show selective outgrowth in mixed cultures grown in broth aerated by continuous shaking, in static broth incubated anaerobically in hydrogen, and on aerobic agar plates, i.e., under conditions not allowing an advantage from pellicle formation. The outgrowth of fim(+) bacteria in aerobic static broth was prevented by the addition of alpha-methylmannoside, a substance that inhibits the adhesive and early pellicle-forming properties of bacteria with type 1 fimbriae. A motile flagellate (fla(+)) variant of a fim(-)fla(-) strain of S. typhimurium outgrew its parent strain in mixed cultures in aerobic static broth, but the selective advantage conferred by motility was weaker than that conferred by fimbriation.     

Adhesion of Salmonella dublin to HEp2 epithelial cells

Two strains of Salmonella dublin , grown serially in brain heart infusion broth, were motile and produced mannose sensitive (MS) but not mannose resistant (MR) haemagglutinins; grown on phosphate buffered agar, the strains were poorly motile and phenotypically MSHA^- MRHA ⁺. In adhesion tests with HEp2 epithelial cells, broth grown bacteria that were motile and MSHA⁺ MRHA^- adhered better than agar grown bacteria that were poorly motile and MSHA^- MRHA⁺. Thus, in adhesion tests with HEp2 epithelial cells, strains of S. dublin behaved like S. typhimurium strains in that their HEp2 cell adhesiveness was associated with motility and production of MSHA.     

Temperature-dependent utilization of meso-inositol: a useful biotyping marker in the genealogy of Salmonella typhimurium

Salmonella typhimurium strains from natural sources either ferment or do not ferment meso-inositol in peptone water in 24 hr at 37 C. Ninety-five percent of the strains that are designated inositol-nonfermenting on the basis of their phenotype at 37 C ferment inositol when incubated at 25 C. Two classes of temperature-sensitive mutants were detected among the 712 strains of S. typhimurium examined. The occurrence of low-temperature fermentation of inositol among wild-type strains of S. typhimurium from biotypes 10 through 13 and FIRN suggested a genealogical relationship between these two groups, and that FIRN strains (fim(-)inl(ts)rha(-)) might have descended from ancestral types like biotype 10 through 13 strains (fim(+)inl(ts)rha(+)).     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

The attachment to, and invasion of HeLa cells by Salmonella typhimurium: the contribution of mannose-sensitive and mannose-resistant haemagglutinating activities

The association of the haemagglutinating activities of Salmonella typhimurium cultures with bacterial adhesion to HeLa cells, and the internalization of the bacteria by HeLa cells, was studied. Adhesion was not inhibited by alpha-methyl-D-mannoside (i.e. adhesion was mannose-resistant), and only four of the six strains tested produced type 1 fimbriae and the associated mannose-sensitive haemagglutinin (MSHA). The other two strains belonged to the non-fimbriate FIRN biogroup. Cultures of all six strains contained a mannose-resistant haemagglutinating (MRHA) activity when grown at 37 degrees C, but cultures of only one fimbriate and one non-fimbriate strain did so when grown at 18 degrees C. From the comparison of cultures grown at 18 degrees C and 37 degrees C, and of mutant strains with the phenotypes MRHA-negative/MSHA-positive, or MRHA-positive/MSHA-negative, it was concluded that the MRHA activity was responsible for the attachment of salmonellae to HeLa cells. Only bacterial adhesion that was resistant to mannose resulted in the internalization of the bacteria by the HeLa cells.     

Adhesion of fimbriated nitrogen-fixing enteric bacteria to roots of grasses and cereals

Summary The role of fimbriae in enterobacterial adhesion to roots of grasses and cereals is discussed. All nitrogen-fixing enteric bacteria isolated in Finland had fimbriae. AllEnterobacter isolates had mannose-binding type-1 fimbriae, whereas most of theKlebsiella isolates had both type-1 and type-3 fimbriae. The strains were isolated from a total of ten different grass species, and no specific association was found between grass species and bacterial fimbriation, biogroup or serogroup. Purified, radiolabeled fimbriae bound to roots ofPoa pratensis in vitro, and bacterial adhesion was inhibited by Fab fragments specific for fimbriae.Klebsiella strains carrying type-3 fimbriae adhered to roots of various grasses and cereals more efficiently than type-1- or nonfimbriated strains, and it was concluded that type-3 fimbriae are the major adhesions ofKlebsiella. Immunofluorescence studies revealed that the bacteria preferentially adhered to root hairs, and to a lesser extent, to the zone of elongation and the root cap mucilage. No strict host specificity in enterobacterial adhesion was observed.     

Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5'-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5'-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47?- W-383) protein was autopolymerized to form filamentous structures of about 80?nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.     

Isolation and Some Properties of Fimbriae of Oral <Emphasis Type="Italic">Streptococcus intermedius</Emphasis>

Streptococcus intermedius 1208-1 carried linear fiber-like fimbriae that extended radially from the cell surface. The fimbriae were isolated by pipetting and sonication and were purified by ammonium sulfate precipitation followed by a column chromatography series. Heat treatment in the presence of sodium dodecyl sulfate resulted in the dissociation into smaller molecules. Rabbit antiserum raised against the purified protein reacted with fimbriae on the surface of bacteria under immunogold staining. Serotype g or g-related strains produced the fimbriae and aggregated in human saliva. The aggregation was inhibited by the anti-fimbriae immunoglobulin Fab fragment or the purified fimbriae.     

d-Ribose Formation by Pseudomonas reptilivora

During the course of some works on sugar metabolism in bacteria, we could find out bacteria having producibility of free d-ribose. Among 1395 strains isolated from soil, only nine strains were found to be able to produce aldopentose which was identified chromato-graphically as ribose. From cultured broth of Pseudomonas reptilivora S-1104, a representative strain among these nine strains, d-ribose was isolated in crystalline form as aldopentose. It was also found that ribose was formed not only from glucose but also from d-fructose, d-arabitol, gluconic acid, etc., and that d-fructose and a glucoside (remained unknown) were also accumulated at the same time in the culture broth of Pseudomonas reptilivora S-1104.     

Electron microscopic observation of Branhamella catarrhalis

The hemagglutination (HA) test was done on 85 strains of Branhamella catarrhalis, isolated from sputum of patients with respiratory infections; 53% were HA positive strains. Three HA positive and three HA negative strains were selected and were observed under the electron microscope. The bacterial cell wall appeared to be lobulated and its total thickness was about 38 nm. The nuclear region consisted of whorls or fibrils and dense bodies. Five strains were fimbriated and one strain was nonfimbriated. The size of fimbriae was about 68 nm in length and 4.5 nm in width. The fimbriae of Branhamella catarrhalis were densely arranged and peritrichous in distribution. There was no change of fimbriation between broth and agar cultures.     

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial anti-serum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.     

Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.     

Role of Mrx fimbriae of Xenorhabdus nematophila in competitive colonization of the nematode host

Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila.     

Biogenesis of Type V pili

Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N-terminal processing of the FimA precursor by arginine-specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease-mediated strand-exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram-negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms.     

The role of motility in the in vitro attachment of Pseudomonas putida PaW8 to wheat roots

The attachment of motile and non-motile strains of Pseudomonas putida PaW8 to sterile wheat roots was assessed in both non-competitive and intra-specific competitive assays. The motile strain showed significantly greater attachment to wheat roots than non-motile strains in phosphate buffer. Overall, the motile strain attached better than the non-motile strain at 10(6), 10(7) and 10(8) cfu ml(-1) in competitive assays and at 10(6) and 10(7) cfu ml(-1) in non-competitive assays. When attachment was studied in Luria broth no significant difference between motile and non-motile strains was detected. P. putida PaW8 cells marked with the luxAB genes were used to compare direct detection of attached cells by luminometry with indirect detection by dilution plate counts following extraction from root material. Although direct detection permitted a rapid assessment (60 s) of attachment to surfaces, dilution plate counts provided a more sensitive method for quantification of bacteria. The detection limits were approximately 10 cfu root(-1) using dilution plate counts compared with 1000 cfu root(-1) using luminometry. All results highlighted the importance of motility for the attachment of P. putida to plant roots in simple model systems. To take this work further, studies to assess the role of motility using complex non-sterile systems are needed.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

小溪自然保护区嗜盐及耐盐菌抗菌活性筛选及生物学特征

以8个敏感菌株(3株革兰阳性菌、3株革兰阴性菌和2株真菌)作为指示菌,采用管碟法对分离自湖南小溪自然保护区普通非盐环境(ordinary non-saline environment)土样中的114株嗜盐及耐盐细菌(含放线菌)进行抗菌活性筛选,并对抗菌活性较强的菌株进行了基于16S rRNA基因序列的系统发育分析和生物学特性研究。结果显示,74株受试菌株的发酵产物具有抗菌活性,阳性率为64.9%。综合分析形态特征、生理生化特征和基于16S rRNA基因序列的系统发育分析数据,表明9株具有较强抗菌活性的菌株分别属于Bacillus属(JSM 081049、JSM 082021-1、JSM 082056、JSM 082080、JSM 082081-1、JSM 082097)、Arthrobacter属(JSM082018)、Brachybacterium属(JSM082044)和Streptomyces属(JSM082030)。     

Occurrence of p-fimbriae in enteropathogenic E coli (EPEC) and in E. coli strains isolated from patients with urinary tract infections]

The aim of this study was to gain knowledge of prevalence of P+ clones among EPEC strains isolated from children with diarrhoea and E. coli strains isolated from urine. Three hundred eighty four E. coli strains isolated from children with diarrhoea were tested. They belonged to 11 serotypes (018, 025, 026, 044, 055, 0111, 0114, 0119, 0124, 0125, and 0128). Nine hundred thirty colonies of E. coli from Mac Conkey's agar plated quantitatively with urine samples of 178 individuals suffering from urinary tract infections were also tested. All strains were assayed by mannose-resistant active haemagglutination test (MRHA) and by slide agglutination using self prepared latex reagent for detection of P fimbriae. Out of 384 E. coli strains tested 122 (31.8%) showed presence of adhesins detected by mannose-resistant active haemagglutination test (MRHA) and in 90 (23.3%) out of all tested strains the presence of P fimbriae was found. The highest percentage of P fimbriae prevalence was found in E. coli belonging to the following serotypes: 018 (in 68.9% strains), 025 (in 29.2% strains), and 0125 (in 25.0% strains). This type of fimbriae was also detected in serotypes 026 (9.1%), 044 (8.7%), 055 (5.6%), and 0119 (in 2 strains out of 5 isolated). Out of 933 colonies of E. coli, isolated from 178 urine samples, 434 (46.5%) colonies gave positive results in MRHA test, including 133 positive in latex test for P fimbriae. These studies showed that for MRHA adhesins, including P fimbriae, a parallel examination of higher number of E. coli was necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

Background  

Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogeniCity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model.