Pectinatus portalensis nov. sp., a relatively fast-growing, coccoidal, novel Pectinatus species isolated from a wastewater treatment plant

The genus Pectinatus is currently composed by two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis , both asociated with beer spoilage. This study describes a novel isolate (strain B6) retrieved from a wastewater treatment plant collecting residues from a large number of wineries. Based on similarity analysis of 16S rRNA gene sequences, strain B6 belongs to the genus Pectinatus . Strain B6 is a strict anaerobe like other Pectinatus species and it presents non-motile, coccoid cells showing a slight oval shape. Strain B6 shows marked physiological differences with other Pectinatus species both in fatty acid composition and carbon source utilization. The most abundant fatty acids found in strain B6 were 18:1 (42.8%) and 16:0 (18.3%) representing a total of over 61% of fatty acids in this microorganism while these fatty acids represented 41.3% in P. cerevisiiphilusT and 2.4% in P. frisingensisT of their total. Fatty acid 15:0 was not significant in strain B6 and represented 28.6% and 13.3% for P. cerevisiiphilusT and P. frisingensisT, respectively. Strain B6 showed a faster growth rate and higher optimum temperature than its relatives P. cerevisiiphilus and P. frisingensis . Strain B6, P. cerevisiiphilus and P. frisingensis could be clearly differentiated by acid production tests from substrates such as esculine and gluconate, and the lack of acid production from rhamnose and fucose among others. G+C mol% content in strain B6 is 36.5%. Based on genotypic and phenotypic differences, strain B6 is proposed as a novel Pectinatus species, P. portalensis nov. sp. Both strain B6 and the two described species of Pectinatus grow on beers and wines. These results provide insights about the origin and reservoirs of Pectinatus species and spoiling alcoholic beverages.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

Heat resistance of Pectinatus sp., a beer spoilage anaerobic bacterium

D. WATIER, I. LEGUERINEL, J.P. HORNEZ, I. CHOWDHURY AND H.C. DUBOURGUIER. 1995. The heat resistance of three strains of Pectinatus (Pectinatus cerevisiiphilus DSM 20466, Pectinatus sp. DSM 20465 and Pectinatus frisingensis ATCC 33332) were measured at different temperatures (50–60°C), different pH (4–6·3), in two media (MRS and wort). Similar D ₆₀ values were obtained for DSM 20465 and ATCC 33332, but DSM 20466 was more resistant in MRS. Moreover the z values were higher in MRS than in wort for the three strains. Thermal destruction at different pH was variable. Results were discussed in relation to factors affecting heat resistance.     

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.     

The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Pseudomonas fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.     

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.     

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

Lipopolysaccharides of anaerobic beer spoilage bacteria of the genus Pectinatus--lipopolysaccharides of a Gram-positive genus

Bacteria of the genus Pectinatus emerged during the seventies as contaminants and spoilage organisms in packaged beer. This genus comprises two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis; both are strict anaerobes. On the basis of genomic properties the genus is placed among low GC Gram-positive bacteria (phylum Firmicutes, class Clostridia, order Clostridiales, family Acidaminococcaceae). Despite this assignment, Pectinatus bacteria possess an outer membrane and lipopolysaccharide (LPS) typical of Gram-negative bacteria. The present review compiles the structural and compositional studies performed on Pectinatus LPS. These lipopolysaccharides exhibit extensive heterogeneity, i.e. several macromolecularly and structurally distinct LPS molecules are produced by each strain. Whereas heterogeneity is a common property in lipopolysaccharides, Pectinatus LPS have been shown to contain exceptional carbohydrate structures, consisting of a fairly conserved core region that carries a large non-repetitive saccharide that probably replaces the O-specific chain. Such structures represent a novel architectural principle of the LPS molecule.     

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni

The organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c.0.2kb, similar to that reported for Campylobacter coli. The 5' flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N-terminal 170 amino acids and C-terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface-exposed regions and may represent areas in which surface epitopes are located.     

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

Nisin, temperature and pH effects on growth and viability of Pectinatus frisingensis, a Gram-negative, strictly anaerobic beer-spoilage bacterium

Pectinatus frisingensis , a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin. An increase in nisin concentration (0 to 1100 IU ml⁻¹) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium. In addition, late exponential cells of P. frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K⁺. Presence of Mg²⁺ up to 15 mmol l⁻¹ did not protect P. frisingensis from nisin-induced loss of viability and K⁺ efflux. Potassium leaks were also measured in P. frisingensis late exponential phase cells exposed to combined effects of nisin addition (100–500 IU ml⁻¹), 10 min mild heat-treatment (50 °C) or rapid cooling (2 °C), and pH (4·0 and 6·2). Net K⁺ efflux from both starving and glucose-metabolizing cells, was more important at pH 6·2, whatever the temperature treatment and nisin addition. Reincubation at 30 °C of P. frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K₊ than heat-treated cells, whatever the pH conditions. A combination of nisin and mild heat-treatment could thus be of interest to prevent P. frisingensis growth in beers.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Modeling growth and off-flavours production of spoiled beer bacteria, Pectinatus frisingensis

The genus Pectinatus includes strictly anaerobic Gram-negative non-spore-forming mesophilic bacteria often referred to as beer-spoilage bacteria. Pectinatus frisingensis was chosen as the reference strain. The organisms were grown in batch cultures under stringent anaerobic conditions in a synthetic medium and with pH regulation. Various glucose and lactate concentrations were used, and a low inoculum reproduced spoilage conditions in bottled beer. Propionate and acetate are the major compounds responsible for the off-flavour of beer. Gompertz curves were fitted to acid-biomass production and glucose consumption; thereby the lag-phase, production rate and final concentrations were derived. Volatile fatty acids production began 19 h after biomass growth. There was no lineareffect of substrate on final concentration of propionate, acetate and biomass. An additive model is proposed for the prediction of bacterial growth and acid production on both glucose and lactate.     

Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola.

The gene coding for the flagellin protein of Roseburia cecicola, an oxygen-intolerant, gram-negative, anaerobic bacterium indigenous to the murine cecum, has been cloned and sequenced. NH2-terminal amino acid sequence data from the flagellin protein were used as a basis for the synthesis of two mixed-sequence deoxyoligonucleotides. The oligonucleotides were used to identify and clone the flagellin structural gene. DNA sequence analysis of M13mp8 and mp9 subclones revealed a protein with a length of 293 amino acids and a molecular weight of 31,370. Comparisons with the sequences of flagellins of other species revealed conserved regions and suggested that although R. cecicola has structural characteristics of a gram-negative bacterium, it may be most closely related to the gram-positive bacteria.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.