Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites.

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

Distribution of the clpX gene in Brachyspira species and reactivity of recombinant Brachyspira pilosicoli ClpX with sera from mice and humans.

Previously, a clpX gene encoding a predicted 67 kDa membrane-associated ATPase subunit of the Clp protease (ClpX) was identified in a porcine strain (95/1,000) of the intestinal spirochaete Brachyspira pilosicoli. In the current study, the distribution of this large clpX gene was investigated in a collection of strains representing all seven Brachyspira spp. Using PCR with internal primers, an 878 bp portion of the gene was detected in 29 of 35 strains (83 %) of B. pilosicoli, 6 of 24 strains (25 %) of Brachyspira hyodysenteriae, 14 of 16 strains (88 %) of Brachyspira intermedia, 6 of 17 strains (35 %) of Brachyspira innocens, 1 of 6 strains (17 %) of Brachyspira murdochii, 1 of 2 strains (50 %) of Brachyspira aalborgi and not in the single strain of Brachyspira alvinipulli. The whole gene was sequenced from 20 Brachyspira spp. strains and compared with the clpX gene from B. pilosicoli 95/1,000 (GenBank accession no. AY466377). The genes had 99.3-99.7 % nucleotide sequence similarity and the predicted products had 99.7-100 % amino acid sequence similarity. The clpX gene from WesB, a human strain of B. pilosicoli, was cloned and expressed as a histidine-tagged fusion protein in Escherichia coli BL21. The purified protein was used to vaccinate mice and their sera were found to recognize the expected approximately 67 kDa protein in whole-cell preparations of WesB. Sera from mice vaccinated with formalin-treated whole-cell proteins of WesB reacted with the recombinant protein. These results indicate that ClpX is both conserved and immunogenic and hence might be useful as a subunit vaccine component for Brachyspira spp. infections. Sera from humans with no known exposure to B. pilosicoli reacted with the recombinant ClpX protein, indicating that it is unlikely to be useful as a reagent for serological detection of Brachyspira spp. infections.

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs.

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.

Reclassification of Serpulina intermedia and Serpulina murdochii in the genus Brachyspira as Brachyspira intermedia comb. nov. and Brachyspira murdochii comb. nov.

A reassessment was undertaken of published characteristics of the five species of anaerobic intestinal spirochaetes in the genus Brachyspira (Brachyspira aalborgi, Brachyspira alvinipulli, Brachyspira hyodysenteriae, Brachyspira innocens and Brachyspira pilosicoli) and the two species in the genus Serpulina (Serpulina intermedia and Serpulina murdochii). Comparisons were made of published descriptions, including phenotypic properties, grouping by multilocus enzyme electrophoresis, DNA base composition, DNA-DNA relative reassociation values and 16S rRNA gene sequence similarity. On the basis of extensive similarities between all the species, it is proposed to reclassify Serpulina intermedia Stanton et al. 1997 and Serpulina murdochii Stanton et al. 1997 in the genus Brachyspira, as Brachyspira intermedia comb. nov. (type strain ATCC 51140T) and Brachyspira murdochii comb. nov. (type strain ATCC 51284T).

Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspiramurdochii.

Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia.

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

The use of multilocus enzyme electrophoresis to characterise intestinal spirochaetes (Brachyspira spp.) colonising hens in commercial flocks.

Multilocus enzyme electrophoresis (MLEE) was used to identify, examine genetic relationships and look at disease associations of a collection of 53 intestinal spirochaete isolates previously recovered from the faeces of adult hens on 14 farms in Qld, Australia. The MLEE results were compared with those previously obtained using species-specific PCR amplifications. The isolates were divided into five Brachyspira species groups by MLEE: Brachyspira murdochii (n=17), B. intermedia (n=15), B. pilosicoli (n=14), B. innocens (n=2) and "B. pulli" (n=1). Three new MLEE groups each containing single isolates also were identified. The results of the PCR assay for B. pilosicoli were concordant with the MLEE results, but the 23S rDNA-based PCR for B. intermedia had failed to detect 8 of the 15 isolates. The B. innocens/B. murdochii nox-based PCR had correctly identified all the isolates of B. murdochii, but did not identify either of the two B. innocens isolates. Using MLEE, isolates from two farms (14%) were identified as B. murdochii, whilst the pathogenic species B. intermedia and B. pilosicoli were present in hens from eight (57%) and five (36%) farms, respectively, and were identified together in four (29%) farms. All seven of the farms with production problems or wet litter were colonised with B. intermedia and/or B. pilosicoli. Six farms had multiple spirochaete isolates available for examination. Two broiler breeder farms both had five isolates of B. pilosicoli that shared the same MLEE electrophoretic type (ET), whilst one laying hen farm had three isolates of B. intermedia that all belonged to the same ET. Hence on each of these farms a predominant strain of a pathogenic species was present. On the other farms isolates of the same species were more diverse and belonged to different ETs. These results show that the epidemiology of intestinal spirochaetal infections in broiler breeder and laying hen flocks can vary considerably between farms, although the reasons for these differences were not established.

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.

The peptidolytic capacity of the spirochete system.

Relatively scant chemical information has been available on the proteinases and peptidases of spirochetes in spite of the association of spirochetes with several serious infections known to plague humans and other animal species. This situation has partly resulted from difficulties in growing some spirochetes under laboratory conditions. The cells of Treponema denticola, a spirochete suggested to be associated with periodontal infections, have turned out to be a good source of new chemical information on those enzymes. Latest studies suggest that the outer cell envelope or the periplasmic space of T. denticola contains several novel proteinases and peptidases (hence called "ectoenzymes") which may contribute to the chronicity of periodontal infections. Some of the oligopeptidases discovered are specific for proline-containing host tissue peptides such as substance P, bradykinin, neurotensin, etc., and possibly small collagen fragments. The only spirochetal peptidases purified to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been obtained from T. denticola. One particular peptidase, suggested to be similar to the oligopeptidase B (EC 3.4.21.83) of Escherichia coli seems to be present in the cell envelope or in the periplasmic space at quite large concentrations. The presence of this and several other peptidases in the outer cell structures of the treponemes suggests that such enzymes are important for the nutrition of these highly motile and invasive organisms. The biological role of these enzymes can thus be envisaged in the peptidolytic processing of host tissue proteins and peptides to gradually smaller molecules to fulfill the nutritional requirements of these organisms. Although the genetic similarity between T. denticola and some other treponemes and spirochetes can be hotly debated, it is nevertheless now possible to use T. denticula enzymes as suitable objects for comparison when the chemistry of other spirochetes is studied.

Genetic characterisation of intestinal spirochaetes and their association with disease.

Multilocus enzyme electrophoresis was used to assess genetic relationships amongst 175 isolates of anaerobic intestinal spirochaetes, including 72 isolates from individuals living in different parts of the world, 102 from pigs and one from a dog. Amongst porcine isolates belonging to the genus Serpulina, a possible new species was identified. All but one of the isolates from man were clustered with the canine isolate and 59 porcine isolates in a distinct group that we have previously called "Anguillina coli". The human and animal spirochaetes in this group had four-to-six axial flagella and most were recovered from individuals with diarrhoea. They included a strain of the so-called "Serpulina jonesii", that was not a true serpulina. These 71 human isolates were distributed into 44 electrophoretic types and had a mean genetic diversity of 0.32. These were further divided into 26 clonal groups. Three of these clones also contained porcine isolates, one of which was strain P43/6/78, the agent of porcine intestinal spirochaetosis. Four of the clones contained human isolates from different sources. One included isolates from Western Australian Aboriginal children and from Italian adults, and the other three contained isolates from Western Australian Aboriginal children and from homosexual males in Sydney, New South Wales. There were no known connections between these human populations. The other spirochaete of human origin was Brachyspira aalborgi, which was distinct from isolates in the genera Serpulina and "Anguillina". Both B. aalborgi and "A. coli" have been associated with human cases of intestinal spirochaetosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[A rapid diagnosis for periodontitis based on enzymatic activity of periodontopathic bacteria. Correlation between enzymatic activity, clinical periodontal parameters, and subgingival level of microorganisms].

There have been various laboratory methods for the microbiological diagnosis of periodontal disease. However, there have been some disadvantages in these methods. In this study of the application of a chair-side test for microbiological diagnosis, the activity of peptidases in periodontal pockets of patients was examined by using the assay system SK-013. SK-013 consists of synthetic substrates and is capable of rapidly (in 15 min) evaluating the activity of specific peptidase from Treponema denticola and Bacteroides species. Using SK-013, we evaluate the correlation between the enzymatic activity, clinical periodontal parameters and subgingival level of microorganisms, including phase contrast microscopy. We calculated the sensitivity and efficacy of SK-013 as a diagnostic indicator in the presence of Spirochetes and in periodontitis. A positive correlation were demonstrated between enzymatic activity, clinical periodontal parameters, the numbers of total cell count, Spirochetes, and M & S ratio. SK-013 was highly sensitive and efficacious (sensitivity: 92%, efficacy: 96%). We concluded that the assay system SK-013 is a useful chair-side method for diagnosing periodontal disease.

Correlation of the hydrolysis of benzoyl-arginine naphthylamide (BANA) by plaque with clinical parameters and subgingival levels of spirochetes in periodontal patients.

Recent studies have shown that the extent of hydrolysis by plaque of the trypsin substrate, N-benzoyl-DL-arginine-2-naphthylamide (BANA), correlates with the numbers and proportions of spirochetes in subgingival plaque samples, and appears to be an indicator of clinical disease. In this study, BANA hydrolysis by subgingival plaque was evaluated in a blind manner for its ability to reflect both clinical parameters and subgingival levels of bacteria and spirochetes. Subgingival plaque samples were collected from periodontally healthy and diseased sites in 23 untreated periodontal patients and in 13 treated and maintained periodontal patients. In untreated patients, BANA hydrolysis was statistically associated with the total number of spirochetes and bacteria in the plaque sample, but in the treated patients BANA hydrolysis was statistically associated only with the spirochetes. Most BANA-positive reactions in both patient groups were from the sites which were clinically diseased and high in spirochetes. The majority of the negative reactions for BANA hydrolysis in both patient groups was among the sites which were periodontally healthy and low in spirochetes. Specificity and sensitivity of the test were above 80% for disease status in untreated patients. The predictive value of a positive and negative test was above 83%. Slightly lower sensitivity, specificity, and predictive values were found in the treated group. The BANA reaction appears to be an accurate and simple indicator of both clinical disease status and plaque levels of spirochetes in individual tooth sites in untreated and treated periodontal patients.

Characteristics of trypsin-like activity in subgingival plaque samples.

Previous studies have demonstrated that the hydrolysis of the trypsin substrate N-benzoyl-DL-arginine-2-naphthylamide (BANA), by subgingival plaque obtained from a single site, correlates best with the numbers and proportions of spirochetes in plaque samples and may serve as an indicator of clinical disease. In this investigation, we determined whether the association between BANA hydrolysis and spirochetes could be obtained in pooled subgingival plaque samples. Concomitantly, the characteristics of this reaction in terms of substrate type and concentration, microbial numbers needed to give a positive reaction as assessed by microscopic counts, rapidity of hydrolysis, and the effect of pH and various additives on the plaque BANA hydrolytic activity have been studied in pooled plaque samples from patients who were periodontally healthy or diseased. In addition, it was determined whether BANA hydrolytic activity found in subgingival plaque reflected contributions from saliva and supragingival plaque. Results indicated that the assay can best be performed with 0.67 mmol/L BANA at pH 7.0. EDTA and CaCl2 gave a slight inhibition and DTT a slight enhancement of the BANA reaction by the pooled plaque suspensions. The majority of the reactions (85%) developed their full color after overnight incubation. BANA hydrolysis was not found in saliva and occurred with much greater frequency in subgingival plaque as opposed to supragingival plaque. Analysis of the data indicated that BANA hydrolysis by pooled subgingival plaque samples is a suitable test for the detection of spirochetes when two or three spirochetes per high microscopic field are present in the sample.

Trypsin-like activity in subgingival plaque. A diagnostic marker for spirochetes and periodontal disease?

Taxonomic screening of subgingival plaque organisms with various enzyme assays have shown that Treponema denticola, Bacteroides gingivalis and an unspeciated Capnocytophaga species possess a trypsin-like enzyme (TLE) that can be detected by the hydrolysis of N-benzoyl-DL-arginine-2-naphthylamide (BANA). As these organisms can be considered to be periodontopathic, it was of interest to determine whether this BANA hydrolyzing enzyme could be detected directly in subgingival plaque samples. Subgingival plaque samples were collected from single sites of known pocket depth, and after dispersal by vortexing, aliquots were incubated overnight with BANA and were counted microscopically. The color reactions were developed with fast garnet, read by the eye and classified as positive (red to red-orange), negative (yellow) and questionable. In the BANA-positive plaques, the spirochetes averaged 43% of the microscopic count, whereas in the BANA negative plaques the spirochetes averaged 8% of the microscopic count. The average pocket depth of BANA-positive plaques was 6.7 mm, whereas the average pocket depth of BANA-negative plaques was 4.5 mm. When both of these parameters were combined, the presence of a positive BANA reaction was usually indicative of subgingival plaques containing greater than 34% spirochetes removed from sites that had probing depths of 7 mm or more. Seventy-one per cent of the plaques removed from untreated periodontal patients were BANA-positive, while only 8% of the plaques removed from successfully treated patients seen at maintenance recall visits were BANA-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

beta-Lactamase production by intestinal spirochaetes.

beta-Lactamase production was demonstrated in four of nineteen strains of intestinal spirochaetes isolated from human subjects. The enzyme was preferentially active against penicillins and was inhibited by clavulanic acid; it was membrane bound and non-inducible. No plasmids were detected in the intestinal spirochaetes and the beta-lactamase-production characteristic was not transferable to non-producing strains.

Isolation and characterisation of intestinal spirochaetes.

Faeces or rectal swabs from 1527 subjects were examined for the presence of intestinal spirochaetes by anaerobic culture on blood agar incorporating spectinomycin (400 mg/l). Twenty three specimens (1.5%) were positive, and only one of these came from a patient with diarrhoea. All positive specimens came from either Asians or known homosexuals. Comparative tests showed a close phenotypic similarity between the human isolates and non-pathogenic porcine intestinal spirochaetes. These organisms differ from Brachyspira aalborgi, a spirochaete isolated from subjects with histologically confirmed intestinal spirochaetosis.

Enzyme activities from eight small-sized oral spirochetes.

The purpose of the present investigation was to detect strains of small-sized oral spirochetes isolated from subgingival plaque for protease, peptidase, lipase, glycosidase, phosphatase, hyaluronidase and chondroitinsulfatase activities. The analyses were routinely carried out with cultures in the early stationary phase of growth after 4 days incubation. Both culture media and harvested spirochete cells were examined for the different enzyme activities. The enzymes were assayed by use of the API ZYM system, by p-nitroanilide derivatized peptides, and by hydrolyzing of mucopolysaccharides incorporated in solid bacterial medium. Relatively strong activities of trypsin-like enzymes, mainly bound to the cells, were observed in all strains. Similarly all strains showed acid phosphatases bound to the cells, too. Extracellular hyaluronidase- and chondroitinsulfatase activities were detected qualitatively in all strains after 7 days growth. The activities of the two mucopolysaccharide degrading enzymes almost disappeared after 10 subcultivations. Weak lipase (butyrate), higher lipase (caprylate), and weak phosphoamidase activities were observed in all cell pellets. No glycosidase activities were found. The observations are discussed by regarding the spirochetal enzymes as potential virulence factors for the development of marginal periodontitis.

Pectinolytic enzymes of oral spirochetes from humans.

Five strains of obligately anaerobic, pectin-fermenting spirochetes were isolated from the subgingival plaque of humans. The strains produced two extracellular enzymatic activities that functioned in pectin degradation. One of these enzymatic activities was pectin methylesterase (EC 3.1.1.11), and the other was pectate lyase (EC 4.2.2.2) of the endo type. The data indicate that the cumulative action of these two enzymatic activities brought about depolymerization of pectin in spirochete cultures. Pectin- or polygalacturonate-degrading hydrolases were not detected. A cell-associated lyase activity that catalyzed polygalacturonate breakdown was present in one of the spirochete strains. In addition to pectin, the isolates utilized polygalacturonic, glucuronic, or galacturonic acid as fermentable substrate but did not neutral sugars, amino acids, or other substrates tested. Although the oral spirochetes did not ferment hyaluronic acid, one of the strains grew in coculture with a hyaluronidase-producing Peptostreptococcus strain in a medium containing hyaluronic acid as fermentable substrate. Two of the isolates were identified as Treponema pectinovorum strains on the basis of their substrate utilization pattern, end products of fermentation, other phenotypic characteristics, and the guanine-plus-cytosine content of their DNA. Even though the pectinolytic isolates were specialized with respect to the fermentable substrates they utilized, they appeared to compete successfully with other microorganisms in their habitat.