Genetic and phenotypic analysis of multicellular behavior in Salmonella typhimurium.
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Bacterial Adhesion↗
Biomedical subjects
U Römling
Publications and source records attributed to U Römling.
The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.
Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.
Arabidopsis Proteins↗
Oxygen tension and nutrient starvation are major signals that regulate agfD promoter activity and expression of the multicellular morphotype in Salmonella typhimurium.
Expression of multicellular behaviour (rdar morphotype) is a characteristic of wild-type Salmonella typhimurium strains. The key target for the regulation of rdar morphotype expression is the agfD promoter. The regulation of two rdar morphotypes, regulated and semi-constitutive (the latter differs from the former by the insertion of A after position -17), by various environmental conditions was studied using transcriptional fusions to the regulated and semi-constitutive agfD promoters by Western blot analysis and phenotypic analysis of the rdar morphotype. AgfD promoter activities were strongly dependent on oxygen tension. Expression maxima were observed in rich medium under microaerophilic conditions and in minimal medium under aerobic conditions. The regulated rdar morphotype was only expressed under conditions of maximal promoter activity. Glucose did not influence rdar morphotype expression, and the two promoters showed no consistent response to pH. In the stationary phase of growth, nitrogen and phosphate depletion were found to be signals that switch on the agfD promoters. In the logarithmic phase of growth, ethanol was the stress signal that enhanced rdar morphotype expression. The results indicate that, although the regulated and semi-constitutive agfD promoters are key factors in the grade of expression of the multicellular behaviour, common signals such as oxygen tension, depletion of nutrients and ethanol vary their levels of expression significantly.
Bacterial Proteins↗
AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.
The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.
Amino Acid Motifs↗
Achieving 100% typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis.
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Bacterial Typing Techniques↗
Flagella modulate the multicellular behavior of Salmonella typhimurium on the community level.
Salmonella typhimurium strains MAE52 and MAE32 display a constitutive multicellular morphotype mediated by the expression of the agfD operon. In those strains, the role of flagella in the formation of various modes of multicellular behavior was investigated. Flagella were not required for the formation of the multicellular morphotype (rdar) on plates. However, visual examination showed that the global behavior of the bacterial community on air-liquid, surface-liquid or cell-cell-liquid interfaces changed in the absence of flagella. No differences in the local cell-cell interactions were observed at the microscopic level. Using Western blot analysis, no co-regulation of flagella and thin aggregative fimbriae, an extracellular component of the multicellular morphotype, was observed either on plates or in standing culture. In a mutant lacking flagella and thin aggregative fimbriae, the contribution of the latter to the multicellular morphotype was dominant. We concluded that independently regulated genes can act in an additive fashion to confer a pronounced multicellular behavior.
Bacterial Adhesion↗
Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.
Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.
Bacterial Proteins↗
Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis.
A comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics. Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence. We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci. Comparison of the physical and genetic maps and determination of the map positions of the strain-specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain-specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome. The two methods were applied to differential genome analysis of clonal divergence in Pseudomonas aeruginosa choosing two clone C isolates from diverse habitats.
Chromosomes, Bacterial↗
Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase.
During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.
Amino Acid Sequence↗
Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.
A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.
Bacterial Adhesion↗
Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr.
The oxidative decarboxylation of coproporphyrinogen III catalysed by an oxygen-dependent oxidase (HemF) and an oxygen-independent dehydrogenase (HemN) is one of the key regulatory points of haem biosynthesis in Pseudomonas aeruginosa. To investigate the oxygen-dependent regulation of hemF and hemN, the corresponding genes were cloned from the P. aeruginosa chromosome. Recognition sequences for the Fnr-type transcriptional regulator Anr were detected -44.5 bp from the 5' end of the hemF mRNA transcript and at an optimal distance of -41.5 bp with respect to the transcriptional start of hemN. An approximately 10-fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr-type regulator, Dnr. Regulation by both proteins required the Anr recognition sequence. Surprisingly, aerobic expression of hemN was dependent only on Anr. An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation. Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN-type enzyme, which was termed HemZ. Expression of hemF was induced approximately 20-fold during anaerobic growth and, as was found for hemN, both Anr and Dnr were required for anaerobic induction. Paradoxically, oxygen is necessary for HemF catalysis, suggesting the existence of an additional physiological function for the P. aeruginosa HemF protein.
Aerobiosis↗
Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation.
Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C) and csgDEFG, from S. typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli. The divergence of the curli region between S. typhimurium and E. coli at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently, S. typhimurium genes on low-copy-number plasmids were able to complement respective E. coli mutants, although not always to wild-type levels. rpoS and ompR are required for transcriptional activation of (at least) the csgD promoter. The high degree of conservation at the protein level and the identical regulation patterns in E. coli and S. typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species.
Bacterial Outer Membrane Proteins↗
Large genome rearrangements discovered by the detailed analysis of 21 Pseudomonas aeruginosa clone C isolates found in environment and disease habitats.
In order to determine primary genetic events which occur during the diversification of a Pseudomonas aeruginosa clone in natural habitats, comparative genome analysis of 21 isolates of a predominant clone, called clone C, derived mainly from patients with cystic fibrosis (CF) and the aquatic environment, was carried out. Physical chromosome maps were constructed for the restriction enzymes SpeI, PacI, SwaI and I-CeuI by one and two-dimensional pulsed-field gel electrophoresis and by comparison with the existing strain C map. The positioning of 26 genes generated the genetic maps. Chromosome size varied between 6345 and 6606 kilobase-pairs (kb). A plasmid of 95 kb was detected in the strains of non-CF origin and, in addition, was found to be integrated into the chromosome of all strains but one CF isolate. Four subgroups of clone C strains were discriminated by the acquisition and loss of large blocks of DNA that could cover more than 10% of the chromosome size. The exchange of DNA blocks which ranged in size from 1 kb to 214 kb occurred preferentially around the terminus of replication region which is poor in biosynthetic genes. Genetic material which was additionally introduced into strain C in comparison with strain PAO seems to be a target of mutational processes in clone C strains. Within and among subgroups CF isolates frequently exhibited large inversions affecting the whole chromosomal structure. We concluded that the exchange of DNA blocks by mechanisms of horizontal transfer and large chromosomal inversions are major factors leading to the divergence of a clone in the species P. aeruginosa.
Chromosome Mapping↗
Large chromosomal inversions occur in Pseudomonas aeruginosa clone C strains isolated from cystic fibrosis patients.
An analysis of the chromosomal structure of 21 Pseudomonas aeruginosa clone C genotypes of various origins was carried out. The circular chromosomes showed various insertions and deletions which did not alter the gene order in 6 environmental and one ear isolate in comparison with strain PAO and strain C. However, the chromosome structure differed by at least one large inversion in 50% of the genotypes (7/14) isolated from cystic fibrosis patients. Recombination endpoints were scattered throughout the chromosome. All but one inversion included the terminus of replication which was displaced by the recombination events. The rearrangements shifted the positions of the rrn operons with respect to the origin of replication, but the transcription of the rrn operons remained in the same direction as replication. The exclusive detection of inversions in isolates from cystic fibrosis lungs, which represent an atypical habitat for P. aeruginosa, supports the theory that peculiar features of this new ecological niche may select, cause or tolerate genomic changes.
Chromosome Inversion↗
Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis.
The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6-23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis.
Adolescent↗
Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations.
In order to determine the genome variability within Pseudomonas stutzeri, 20 strains representing the seven described genomovars and strain JM300 were analyzed by using various resolution levels of rare cutting enzymes. XbaI and SpeI fingerprints revealed a high degree of heterogeneity of restriction patterns that did not correlate with the division into genomovars. However, a fragment pattern comparison led to the establishment of several groups of clonal variants within genomovars. One circular chromosome ranging in size from 3.75 to 4.64 Mb constitutes the genome of P. stutzeri strains. The I-CeuI, PacI, and SwaI low-resolution map of P. stutzeri type strain CCUG 11256 shows the locations of 12 genes, including rrn operons and the origin of replication. I-CeuI digests of the 20 strains studied plus the positions of six genes allowed a comparison of the rrn backbone organization within genomovars; the four rrn operons seemed to be at similar locations with respect to the origin of replication, as did the rest of the genes. However, a comparison of I-CeuI cleavage maps of the genomovar reference strains revealed a diverse genome organization in the genomovars relative to rrn operons and gene locations. In most genomovars, rrn operons are not arranged around the origin of replication but are equally distributed on the chromosome. Strain JM300 does not belong to any described genomovar, as determined from the organization of its genome. Large chromosomal rearrangements seem to be responsible for the differences in superordinate genome structure and must have played an important role in P. stutzeri diversification and niche colonization. An ancestral chromosome is suggested, and some plausible pathways for the generation of the various genome structures are proposed.
Blotting, Southern↗
Epidemiology of chronic Pseudomonas aeruginosa infections in the airways of lung transplant recipients with cystic fibrosis.
BACKGROUND: The source of airway colonisation with Pseudomonas aeruginosa is not well defined in patients with cystic fibrosis after lung transplantation. Using a DNA-based typing system a study was undertaken to investigate whether lung transplant recipients acquired new strains of P aeruginosa or retained those they had before transplantation. METHODS: Seventy four P aeruginosa isolates taken before and after transplantation were analysed from 11 patients with cystic fibrosis who had undergone lung transplantation in the Medical School of Hannover between 1988 and 1994. The genetic relatedness of the 74 P aeruginosa strains was evaluated from macrorestriction fragment pattern similarity. RESULTS: Each of the 11 lung transplant recipients harboured one identical P aeruginosa clone before and after transplantation. The airways of four of the 11 patients were preoperatively colonised by two or three different clones, but six months after transplantation only one clone was detectable. CONCLUSIONS: These results show that there is no change in the P aeruginosa population in the airways of lung transplant recipients before and after transplantation and it is assumed that the chronic drainage of P aeruginosa into the lung allografts is caused by the bacterial reservoir in the paranasal sinuses and the trachea.
Adolescent↗
Infections with Pseudomonas aeruginosa in patients with cystic fibrosis.
The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid, LPS-, pyocin- and phage-deficient, secrete less virulence determinants and shift the production of cytokines evoked in neutrophils. On the other hand, other properties such as antimicrobial susceptibility or adherence to bronchial mucins remain highly variable reflecting the capacity of P. aeruginosa to adapt to ongoing changes in the CF lung habitat.
Cystic Fibrosis↗