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Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid.

This work was performed to establish a model describing bacterial surface structures involved in biofilm development, in curli-overproducing Escherichia coli K-12 strains, at 30 degrees C, and in minimal growth medium. Using a genetic approach, in association with observations of sessile communities by light and electron microscopic techniques, the role of protein surface structures, such as flagella and curli, and saccharidic surface components, such as the E. coli exopolysaccharide, colanic acid, was determined. We show that, in the context of adherent ompR234 strains, (i) flagellar motility is not required for initial adhesion and biofilm development; (ii) both primary adhesion to inert surfaces and development of multilayered cell clusters require curli synthesis; (iii) curli display direct interactions with the substratum and form interbacterial bundles, allowing a cohesive and stable association of cells; and (iv) colanic acid does not appear critical for bacterial adhesion and further biofilm development but contributes to the biofilm architecture and allows for the formation of voluminous biofilms.

Bacterial Adhesion↗

DNA reshaping by MukB. Right-handed knotting, left-handed supercoiling.

MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations.

Acyl Carrier Protein↗

SseG, a virulence protein that targets Salmonella to the Golgi network.

Intracellular replication of the bacterial pathogen Salmonella enterica occurs in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Maturation of the SCV has been shown to occur by selective interactions with the endocytic pathway. We show here that after invasion of epithelial cells and migration to a perinuclear location, the majority of SCVs become surrounded by membranes of the Golgi network. This process is dependent on the Salmonella pathogenicity island 2 type III secretion system effector SseG. In infected cells, SseG was associated with the SCV and peripheral punctate structures. Only bacterial cells closely associated with the Golgi network were able to multiply; furthermore, mutation of sseG or disruption of the Golgi network inhibited intracellular bacterial growth. When expressed in epithelial cells, SseG co-localized extensively with markers of the trans-Golgi network. We identify a Golgi-targeting domain within SseG, and other regions of the protein that are required for localization of bacteria to the Golgi network. Therefore, replication of Salmonella in epithelial cells is dependent on simultaneous and selective interactions with both endocytic and secretory pathways.

Amino Acid Sequence↗

Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes.

A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.

Bacteria↗

Capsule shields the function of short bacterial adhesins.

Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins. This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms.

Adhesins, Bacterial↗

Aztreonam: the first monobactam.

A novel screening procedure led to isolation of the structurally unique, bacterially produced, monocyclic beta-lactam antibiotics early in 1979. These naturally occurring "monobactams" were not clinically useful as antibiotics because of their poor antibacterial properties. They were, however, found to interact with certain penicillin-binding proteins of bacteria and thus to interfere with the biosynthesis of bacterial cell walls. The focus of monobactam development then turned toward increasing the binding activity of the beta-lactam ring of the molecule. Aztreonam was the first compound to emerge that fulfilled the objectives of the program. It is relatively inactive against gram-positive and anaerobic bacteria but is extremely effective against aerobic gram-negative bacteria, even in low concentrations. In addition, it is highly resistant to enzymatic hydrolysis by beta-lactamases and demonstrates a high degree of stability against plasmid-mediated gram-negative lactamases. With the chromosomally mediated beta-lactamases, on the other hand, aztreonam can act either as an inhibitor or as a poor substrate. It is unique in that it does not induce production of chromosomally mediated enzymes. Interference with normal gut flora by the use of broad-spectrum antibiotics can result in decreased defense capacity and can lead to intestinal colonization by resistant pathogenic organisms. Therapy directed specifically against the invading pathogen is thus preferred. Such directed therapy is provided by aztreonam. Its narrow spectrum can, if necessary, be broadened by combining it with other antibiotics while continuing to maintain an alternative to the more generalized "shotgun" therapy with its attendant side effects such as disturbances of the natural gut flora, diarrhea, and the emergence of resistant bacteria.

Animals↗

Comparison of bacterial populations and chemical composition of dairy wastewater held in circulated and stagnant lagoons.

AIMS: This study compared the chemical, physical and bacterial composition of circulated and stagnant dairy wastewaters. METHODS AND RESULTS: Samples taken from circulated and stagnant wastewater lagoons, over a 1-year period, were analysed for 10 chemical (total N, NH3, NO3, NO2, Na, Ca, HCO3, Fe, P and K) and six physical (biological oxygen demand, chemical oxygen demand, dissolved solids, electrical conductivity, pH and sodium absorption ratio) parameters and were found to be similar. The 16S rDNA genes from the samples were amplified, cloned and BLAST analysed. In total, 996 stagnant and 1052 circulated wastewater derived sequences were obtained, comprising 294 and 362 operational taxonomic units (OTUs) from the circulated and stagnant wastewaters respectively. Coverage estimates of the OTUs identified were 72.1% for the stagnant, and 63.6% for the circulated wastewater libraries. The greatest difference between the two wastewaters was a c. sixfold greater number of sequences representative of the family Chromatiaceae in the circulated wastewater derived library and a c. fivefold greater number of sequences representative of the phylum Chloroflexi in the stagnant wastewater derived library. CONCLUSIONS: Circulation of dairy wastewater does not affect any of the chemical or physical parameters tested; however, circulation does alter the bacterial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that circulation of dairy wastewater promotes the growth of bacteria within the family Chromatiaceae and that stagnant systems promote the growth of the phylum Chloroflexi.

Absorption↗

rpoB-PCR amplified gene and temporal temperature gradient gel electrophoresis: a rapid tool to analyse bacterial strains representative of cold-smoked salmon microflora.

AIM: To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. METHODS AND RESULTS: DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. CONCLUSION: The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis.

Animals↗

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.

Bacteria↗

[Theory and practice of macrorestriction analysis for the clonal analysis of pathogens].

The macrorestriction analysis of bacterial genomes is a method that is generally applicable to the typing of bacteria. The chromosome is cleaved with a restriction endonuclease that cuts infrequently and subsequently separated by pulsed-field gel electrophoresis. The fragment pattern defines the genotype of the strain. The relatedness of strains is evaluated from the similarity of the fragment patterns. The quantitative comparison of fragment patterns allows the definition of the infrasubspecific rank of a clone. Macrorestriction analysis is a sensitive and specific method to trace the origin and spread of infections and to analyse the clonal structure of bacterial populations.

Base Sequence↗

Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste.

Microbial communities were characterized at contaminated sites that had elevated levels of nitrate, nickel, aluminum, and uranium (up to 690 mM, 310 microM, 42 mM, and 30 microM, respectively). The bacterial community structure based upon clonal libraries of the SSU rRNA genes (screened clones = 876) was diverse at the background site, but the three acidic samples had decreased diversity and the majority of clones were closely related to Azoarcus and Pseudomonas species. Arthrobacter and Novosphingobium sequences were recovered from the background samples but not the acidic sites, and similar pseudomonad populations were present at the background and acidic sites albeit at different relative abundances. Heterologous sequence coverage analyses indicated the microbial communities at the contaminated sites were very similar (p = 0.001) but different from the background site. Bacterial isolates (n = 67) classified as beta-or gamma-Proteobacteria, high G+C Gram-positive or low G+C Gram-positive were obtained from the background and one contaminated sample, and some of the isolates had less than 95% sequence identity with previously observed microorganisms. Despite variations in nitrate and heavy metal levels and different proximities to the source ponds, the three acidic samples had similar microbial populations. However, the least contaminated site (lowest nitrate and aluminum) had increased diversity compared to the other acidic samples. The results suggested that the combined contamination has decreased the microbial diversity, and Azoarcus populations were observed at a drastically increased frequency compared to the background site that had a more even distribution of multiple taxa.

Aluminum↗

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls.

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH2CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)39. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling. The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.

Bacillus subtilis↗

Change of microbial community structure by respiratory quinone profile at intermittently aerated membrane bioreactor.

In the present study, the microbial community structure in an intermittently aerated submerged membrane bioreactor treating domestic wastewater was observed using the respiratory quinone profiles. The effects of different time interval for the aerobic and anoxic period on microbial community structure were examined with 60/90 min (Step 1) and 90/60 min (Step 2) as anoxic/aerobic periods. There was an observable slight difference in microbial community structure between Step 1 and Step 2 in the submerged membrane bioreactor. The dominant quinone types for Step 1 at both anoxic and aerobic conditions were UQ-8 followed by UQ-10 and MK-6, but those for Step 2 were UQ-8, MK-6 and MK-10(H4). The microbial diversity of Step 1 and Step 2 based on the composition of all quinones was 10.6-11.7 and 13.3-13.0 for anoxic and aerobic conditions, respectively. The present results suggest that the introduction of intermittent aeration into the submerged membrane bioreactor has little influence on the bacterial community structure.

Air Movements↗

Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization.

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery. In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations. Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films. Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium). The inhibition of E. coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture. Noticeably, the biofunctionalization could be achieved only when positively charged poly(l-lysine) was the outermost layer of the film. On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure. These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial. The modified surfaces are active against microbial infection and represent a novel means of local host protection.

Adsorption↗

Influence of flavomycin on ruminal fermentation and microbial populations in sheep.

Flavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin day(-1) decreased ruminal ammonia and total volatile fatty acid concentrations (P<0.001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some high-activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.

Ammonia↗

[Revelation and phylogenetic analysis of the predominant bacterial community associated with sponges in the South China Sea based on PCR- DGGE fingerprints].

The predominant bacterial community structure of Dysidea avara and Craniella australiensis in the South China Sea were revealed by PCR- DGGE fingerprinting in the present study. With further cloning, sequencing and phylogenetic analysis, it was found that Proteobacteria predominated in these two sponges. Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria were found in Dysidea avara and only Gammaproteobacteria found in Craniella australiensis. Although Bacteroidetes were found in both sponges, they differed in the species. These bacteria were found in sponges firstly. The bacteria in Craniella australiensis show more complex diversity than that in Dysidea avara. Because compared with Dysidea avara, Craniella australiensis include Actinobacteria, Firmicutes, etc. The bacterial community diversity in these two sponges indicates that the sponge-associated bacteria are host-specific even if the hosts are from the same marine location. DGGE fingerprint-based analysis should integrate with band cloning and sequencing, phylogenetic analysis, etc., molecular techniques to get precise results for the microbial community and diversity revelation. The research of studying sponge microbe by DGGE technique is initial work, that will accelerate the development of sponge microorganisms item.

Animals↗

Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm.

A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia-oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%.

Bacteria↗

[Dynamics of enterobacterial multiplication in a mouse model of intraperitoneal infection in the presence of iron cations as dependent on the availability and type of K antigens].

In this work the results of research on the influence of iron cations on the characteristics of the infectious process caused by different enterobacteria (Shigella sonnei, Shigella flexneri, Escherichia coli) are analyzed. In the experimental intraperitoneal infection of mice in the presence of saccharose and iron cations S. sonnei in phase I showed the decrease of 1g LD50 by 3-4 orders, while S. flexneri in the S-form, by not more than 1 order. The absence of correlation between the virulence of the Shigella species used for comparison, as determined in the keratoconjunctival test, and their behavior in vitro in the presence of iron was revealed. E. coli reference strains synthetizing (according to the nomenclature of I. Orskov et al.) "true" K-antigens (K1, K10) or "not true" ones (K8, K9, K27, K57) also showed different virulence in the experimental infection used in this research: the behavior of the former group corresponded to that of S. sonnei in phase I, the latter group occupied the intermediate position between the former group and S. sonnei on one hand and S. flexneri on the other hand. The sharp drop of 1g LD50 after the injection of S. sonnei in phase I in combination with iron cations can be attributed to differences in the characteristics of bacterial surface structures with antiphagocytic function and indicates that the species-specific antigen of S. sonnei in phase I should be classified with K-antigens. The experimental intraperitoneal infection of mice in the presence of trivalent iron cations can be used for making a tentative judgement on the presence of K-antigens in enterobacteria.

Animals↗