The ecology of Cytophaga–Flavobacteria in aquatic environments

Culture-dependent and -independent studies have found that prokaryotic assemblages are quite diverse in aquatic habitats and contain representatives of virtually all of the roughly 40 divisions of bacteria and the major archaeal groups found so far in the biosphere [1]. Fortunately, not all of these prokaryotic groups are abundant in the plankton nor important in all biogeochemical cycles. Autotrophic and heterotrophic bacteria dominate the prokaryotic biomass in surface waters, as Archaea appear to be abundant only in the plankton of the deep oceans [2]. Among the heterotrophic bacteria, the two most abundant groups are often the Proteobacteria and the subject of this review, the Cytophaga–Flavobacteria cluster (Table 1). The relative abundance of the major heterotrophic bacterial groups in aquatic ecosystems, as determined by FISH with oligonucleotide probes The table includes only studies using probes for at least three proteobacterial subdivisions (alpha, beta and gamma) and for the Cytophaga–Flavobacteria cluster (CF). Ranges are given when the study reports several values taken over time or within a habitat. When more than depth was sampled, the surface value is reported unless noted otherwise. All DAPI-positive microbes, probably nearly all bacteria with a few Archaea and fewer (to none) Eukarya. Percent of DAPI-positive microbes that are recognized by the Eub338 probe. The relative abundance of the major heterotrophic bacterial groups in aquatic ecosystems, as determined by FISH with oligonucleotide probes The table includes only studies using probes for at least three proteobacterial subdivisions (alpha, beta and gamma) and for the Cytophaga–Flavobacteria cluster (CF). Ranges are given when the study reports several values taken over time or within a habitat. When more than depth was sampled, the surface value is reported unless noted otherwise. All DAPI-positive microbes, probably nearly all bacteria with a few Archaea and fewer (to none) Eukarya. Percent of DAPI-positive microbes that are recognized by the Eub338 probe. This paper reviews recent studies that have applied molecular methods to examine uncultured Cytophaga–Flavobacteria in freshwaters and the oceans, with the ultimate goal of using this information to better understand the role of heterotrophic bacteria in carbon cycles and other biogeochemical processes. The importance of heterotrophic bacteria in biogeochemical processes is now well appreciated, but until recently geochemists and field-orientated microbial ecologists considered these microbes as if they were a single group, even though microbiologists have been accumulating for several years information about the taxonomic and phylogenetic make up (‘community structure’) of heterotrophic bacterial communities. Only recently, however, have microbial ecologists been able to link community structure with specific biogeochemical processes (‘function’) [3]. Here I summarize briefly our progress in these areas while discussing what we know about Cytophaga–Flavobacteria in aquatic habitats. This microbial group is a natural starting point because of its high abundance in many freshwater and marine systems. The Cytophaga–Flavobacteria cluster belongs to a diverse bacterial division that has been labeled differently over the years. Hugenholtz et al. [1] and other authors have used ‘Cytophagales’ while Hamana and Nakagawa [4] recently stated that the division name was ‘Flavobacterium–Flexibacter–Bacteriodes–Cytophaga’. The latter name does give some indication of the diversity of this division and highlights some of the major genera (Fig. 1). The label used in the most recent edition of Bergey's Manual of Systematic Bacteriology [5] is simply ‘Bacteroidetes’, which will be used in this review. A more common label, however, is Cytophaga–Flavobacteria–Bacteriodes (or Flavobacterium–Bacteriodes–Cytophaga and other permutations), similar to the label (Cytophaga–Flexibacter–Bacteriodes) originally used by Woese and colleagues [6]. Other genera, which are prominent because of their association with humans, include Prevotella and Porphyromonas[7]. A phylogenetic tree of the Bacteroidetes division, illustrating the Cytophaga–Flavobacteria cluster, based on nearly complete 16S rRNA sequences. The tree was constructed using ARB and the latest data set (May 2001) available at http://www.arb-home.de/. The numbers refer to the number of strains grouped together. The Cytophaga–Flavobacteria cluster can be characterized by both phenotypic traits and 16S rRNA sequence data. As first described by Winogradsky, Cytophaga-like bacteria are unicellular, gliding, nonspore-forming Gram-negative rods, although shape can vary [8]. Colonies of many Cytophaga-like bacteria are pigmented due to flexirubin-type pigments found only in these bacteria and flavobacteria. Most notably, Cytophaga–Flavobacteria are chemoorganotrophic and are especially proficient in degrading various biopolymers such as cellulose, chitin, and pectin [8] (see also references in [9]). Another distinguishing feature is that Cytophaga–Flavobacteria are mainly aerobic, although some anaerobic strains are currently classified as Cytophaga[8]. In contrast, the bacteria within the Bacteroides subgroup are all anaerobes and are found most notably in the microflora of the human colon, making up 30% of total fecal isolates (see references in [9]). In his early review of bacterial systematics, Woese [10] did not use the term ‘Cytophaga–Flavobacteria cluster’, but he did divide 19 16S rRNA sequences from Bacteroidetes bacteria into two clusters, the Bacteroides and the Flavobacterium subdivisions. The number of Bacteroidetes sequences has increased by over 10-fold since Woese's review, so there are now several other groups in the Bacteroidetes division, but most of the Cytophaga and Flavobacteria species still cluster together (Fig. 1). However, one sign of problems in Bacteroidetes systematics is that the genus name Cytophaga is scattered throughout the entire Bacteroidetes phylogenetic tree, as illustrated by the tree presented recently by Weller et al. [7]. It seems that the call by Nakagaw and Yamasato [11] to divide Cytophaga into several genera and higher taxa has not been fully heeded. The large diversity in rRNA sequences complicates several molecular methods for examining members of Bacteroidetes and other bacterial groups, but its impact on one method, fluorescence in situ hybridization (FISH) with oligonucleotide probes, is critical for this review. By far the most widely used FISH probe for Cytophaga–Flavobacteria is CF319a [9]. Although often termed a probe for Cytophaga–Flavobacteria, CF319a also hybridizes to some members of the Bacteroides–Prevotella cluster [7]. What is also noteworthy is that CF319a does not recognize several Cytophaga–Flavobacteria[7], implying that studies using this probe have underestimated Cytophaga–Flavobacteria abundance. One strain not recognized by the CF319a probe is Cytophaga hutchinsonii; the genome of this bacterium is now being sequenced by the Joint Genomic Institute (http://www.jgi.doe.gov/). Even though CF319a misses several Bacteroidetes genera, it remains the most general probe for the Bacteroidetes division [7]. Rather than replacements for CF319a, new probes are needed to examine clusters within the Bacteroidetes division, including those recognized by the CF319a probe. Weller et al. [7] suggested some new probes, but these need to be applied to environmental samples. Eilers et al. [12] used new probes to count culturable Cytophaga–Flavobacteria in the North Sea; the FISH data indicated that these strains were about 6% of total prokaryote abundance (DAPI counts) in April through September, which is a large fraction for a single bacterial lineage. Weller et al. [7] mentioned that they were unable to design habitat-specific probes that recognize bacteria found uniquely in freshwaters, the oceans, or soils. Bacteria in the Bacteroidetes division can be found in just about every habitat in the biosphere, including kusaya [13] (a Japanese delicacy consisting of putrid fish), rumens [14], hydrothermal vents [15], rocks and sea-ice in Antarctica [16], and sediments of lakes and the oceans [17]. Cytophaga–Flavobacteria seem particularly common in the oceans, according to FISH. In fact, in many oceanic habitats, the Cytophaga–Flavobacteria cluster is the most abundant of all bacterial groups (Fig. 2), accounting for as much as half of all bacteria potentially identified by FISH. Generally, β-proteobacteria are the most abundant group in freshwater systems (Table 1), although Cytophaga–Flavobacteria can dominate mature freshwater biofilms [18], and they were abundant in two Japanese rivers [19] and a river-reservoir system in Spain [20]. Cytophaga–Flavobacteria can be abundant in sediments [21], and in one study of Wadden Sea sediments, CF319a-positive bacteria were the most abundant group even in anoxic layers [17]. Summary of FISH results for the Baltic [72], North Sea [27], California [24], and the Antarctic circumpolar current in the Southern Ocean [74]. The Greek letters indicate the subdivisions of proteobacteria and ‘CF’ is Cytophaga–Flavobacteria. See Table 1 for other environments. The error on FISH data is on the order of 20%. In contrast to the FISH results, few Cytophaga–Flavobacteria sequences have been found by most studies of clone libraries of 16S rRNA genes from free-living bacterial assemblages. In one review of these studies, Giovonnoni and Rappé[22] noted that clone libraries from marine systems were dominated by the following heterotrophs, in descending order: three groups of α-proteobacteria (SAR11, Roseobacter, and SAR116); a γ-proteobacterial group (SAR86); and Actinobacteria. The Cytophaga–Flavobacteria cluster was not in this list. Several Cytophaga–Flavobacteria sequences have been retrieved from particulate detritus communities [23] but few from free-living microbial assemblages, which usually make up >90% of total microbial abundance in the oceans. There are two possible explanations for the difference between the FISH and clone library results. One is simply that all clone libraries to date have been constructed from sites or at times when Cytophaga–Flavobacteria were relatively sparse. After all, bacterial community structure varies greatly among habitats and over time. Another explanation is that clone-library construction is biased against the Cytophaga–Flavobacteria cluster. This latter explanation appears to the case. Two studies have compared FISH and clone libraries of 16S rRNA genes from the same water sample. FISH results indicated that the Cytophaga–Flavobacteria cluster was the most abundant bacterial group along a transect off the coast of California [24], although a few stations had large numbers of α-proteobacteria. In contrast to the FISH results, libraries from two sites of this transect were dominated by 16S rRNA genes from α-proteobacteria, and only five of 169 clones were from Cytophaga–Flavobacteria. Similar to other studies [22], the α-proteobacterial group SAR 11 was abundant in one of the libraries (12% of all clones), but SAR 11-like bacteria were <1% of total bacterial abundance according to FISH. Clones from the γ-proteobacterial group SAR 86, which has attracted much attention recently [25,26], were also abundant in the libraries (about 10%), but these bacteria averaged only 2% of total abundance according to FISH. Eilers et al. [27] also found discrepancies between FISH and clone-library results. Specifically, these authors did not find any 16S rRNA Cytophaga–Flavobacteria clones in a library of 54 clones from a North Sea sample, yet this bacterial group was the most abundant one as identified by FISH. In short, the Cytophaga–Flavobacteria cluster is under-represented and the α-proteobacteria over-represented in clone libraries, compared with the FISH data (Fig. 3). Frequency of clones from various bacterial groups in libraries of 16S rRNA genes compared with their relative abundance as estimated by FISH in California coastal waters. The Greek letters indicate the subdivisions of proteobacteria and ‘CF’ is Cytophaga–Flavobacteria. Data from Cottrell and Kirchman [24]. Of course, FISH is not perfect [28]. One problem is that a relatively large fraction (ca. 50%) of the prokaryotic cells sometimes cannot be probed with any oligonucleotide probe, including the Eub338 probe, which should recognize all bacteria (but see reference [29]). However, Cottrell and Kirchman [24] argued that, for their samples, the composition of clone libraries would still overestimate the α-proteobacterial abundance as determined by FISH, even if all bacteria not detected by FISH were α-proteobacteria. Nonspecific hybridization with CF319a to proteobacteria, which would lead to overestimating Cytophaga–Flavobacteria abundance, has not been observed to date (R. Amann, pers. comm.). On the contrary, it seems likely that CF319a misses members of the Cytophaga–Flavobacteria cluster (see above), thus leading to underestimates of their numbers. The PCR step is an obvious place to look for biases against the Cytophaga–Flavobacteria cluster. Suzuki et al. [30] pointed to the mismatches between EubA (the reverse primer used by Cottrell and Kirchman [24,47]) and the 16S rRNA sequences from their Bacteroidetes clones. However, these mismatches seem insufficient to explain the discrepancy, because clone libraries made with other primer pairs also have few Cytophaga–Flavobacteria 16S rRNA genes [24]. For example, a library of 190 clones made with primers 27F and LSU1933R had only three Bacteroidetes clones compared with 67 from α-proteobacteria [30]. The library of Eilers et al. [27] made with primers 8f and 1542r had four α-proteobacterial clones (and 49 from the γ-proteobacterial subdivision) but none from the Bacteroidetes division. It seems unlikely that primer mismatches explain the paucity of Bacteroidetes clones in all of these libraries. Biases other than primer mismatches need to be considered. Regardless of the explanation, relatively few 16S rRNA sequences from the Cytophaga–Flavobacteria cluster are in the databases. According to the RDP database (http://www.cme.msu.edu/rdp; June 2000 release), the entire Bacteroidetes division is represented by 781 sequences or about 5% of all 16S rRNA sequences, almost an order of magnitude less than the nearly 7000 sequences from the Proteobacteria division. The discrepancy is even larger when sequences of only uncultured bacteria are considered. A search of GenBank (May 2001) turned up 93 16S rRNA gene sequences when ‘Cytophaga’ and ‘uncultured’ were used as keywords. An analogous search for uncultured proteobacterial sequences yielded over 1700 entries. Glöckner et al. [31] reported new Cytophaga–Flavobacteria 16S rRNA sequences from several freshwater habitats, but more sequence data are still needed. Heterotrophic bacteria are the dominant organisms in aquatic habitats using dissolved organic material (DOM), which is one of the largest of organic carbon in the biosphere, and thus is critical in carbon and cycles. of is the first step in the microbial that about of ecologists in carbon would to know the role of various bacterial groups in up The Cytophaga–Flavobacteria cluster is one obvious starting point because of its high abundance, but also because these bacteria have a role in and isolates of Cytophaga–Flavobacteria are well to be proficient in degrading biopolymers such as and chitin, of the high molecular fraction of This fraction is being by organic geochemists because and of are especially in the oceans Two of indicate that Cytophaga–Flavobacteria have a role in using in aquatic not just in several studies have that Cytophaga–Flavobacteria are on particulate organic detritus in marine habitats In contrast, detritus in the and in was dominated by although Cytophaga–Flavobacteria more abundant over time on organic made from as well as marine One major phenotypic of Cytophaga–Flavobacteria, use of organic would explain the of these bacteria on both particulate detritus and in free-living assemblages. It be argued that the high abundance of Cytophaga–Flavobacteria in the free-living is not due to use of organic material (or any but the of these bacteria from communities. However, and of particulate detritus are usually much less than those of in most aquatic habitats, to the Cytophaga–Flavobacteria biomass observed at least in the oceans (Table 1). microbial communities Cytophaga–Flavobacteria, but these cells be able to in the free-living community in order to in their high abundance. the that similar of Cytophaga–Flavobacteria are found free-living and communities can be by use of organic available to both that organic than between these two communities. The detritus by studies was material by or material on with large is about the microbes with other of such as and The between the free-living community and microbes with these is probably not The of for use by uncultured Cytophaga–Flavobacteria from a new that and FISH a of this Cottrell and Kirchman of and and of these two biopolymers by bacterial assemblages in coastal of Bacteria to the Cytophaga–Flavobacteria cluster were the most abundant microbes using and (Fig. but few of these bacteria were among the microbes using although a large fraction using were also Cytophaga–Flavobacteria. The data that was dominated by α-proteobacteria in these coastal waters. with these results, et al. found when were dominated by α-proteobacteria, but of when α-proteobacteria and the abundance of Cytophaga–Flavobacteria and β-proteobacteria of and by bacterial groups in coastal waters, as determined by The Greek letters indicate the subdivisions of The labeled with those bacteria not identified by any of the Data taken from Cottrell and Kirchman The error on FISH data is on the order of 20%. data not indicate that Cytophaga–Flavobacteria are of using and other larger than cannot be even bacteria need to be able to up or from the of However, bacteria be less proficient than other bacteria in using molecular of molecular of are high to the need for bacteria to systems for by bacteria with systems is probably not detected by the method, which is to the most Another for the role of Cytophaga–Flavobacteria in using would be to the abundance of specific bacterial groups times or when the of is such as A few studies have to Cytophaga–Flavobacteria abundance in marine and freshwater habitats but it is to any general with these data. Eilers et al. [12] did large in Cytophaga–Flavobacteria in the North Sea; this bacterial group was the most abundant through most the but other bacterial groups also increased when Cytophaga–Flavobacteria it is to that Cytophaga–Flavobacteria more than other bacteria when biomass was to anoxic sediments and Cytophaga–Flavobacteria increased to in a following a that in organic material Of course, other than will the of Cytophaga–Flavobacteria and other bacteria in natural environments. The in uncultured Cytophaga–Flavobacteria, in the has not been by on by Cytophaga–Flavobacteria was by studies over years (see review by and other studies of to ecologists are nearly as current seems One specific is the paucity of sequence data on Even though by Cytophaga–Flavobacteria is well GenBank has only three and sequences from the entire Bacteroidetes over of the sequences are from bacteria while the sequences are mainly from The genome of has found several genes for but more sequence data are needed to the and of these Data on are in examining the and biogeochemical role of bacterial groups in natural environments. data explain the abundance of Cytophaga–Flavobacteria in some environments. One of the studies with data is that of et al. authors used FISH to over time the abundance of several bacterial groups of water through a Even though and were not bacterial abundance increased in these because the the a major in et al. found that the of Cytophaga–Flavobacteria was nearly than that of the other bacterial groups they (Fig. are some that the high abundance of Cytophaga–Flavobacteria is due to high of bacterial groups in to the of all DAPI-positive is the of the bacteria recognized by the Eub338 probe. The Greek letters indicate the subdivisions of Data taken from et al. However, other indicate that Cytophaga–Flavobacteria not than other In similar to those by et but with et al. found that Cytophaga–Flavobacteria recognized by the CF319a the in a in although only more than β-proteobacteria recognized by the probe. A subgroup of β-proteobacteria the In with from the and et al. found that both and α-proteobacteria than Cytophaga–Flavobacteria in in which microbes were 10-fold with The in the by et al. were with data. et al. found that the proteobacterial subdivisions dominated the cells by cells in the cells to the Cytophaga–Flavobacteria cluster. et al. used to of three groups of bacteria by authors found that the group dominated by Cytophaga–Flavobacteria had the in the of the of the three groups were similar in the and deep The abundance of the total bacterial community and of specific bacterial groups is relatively over time time in most aquatic habitats, because of these microbes is by due to both and Generally, for most of bacterial but the impact of and on bacterial groups is of Cytophaga–Flavobacteria have been in but is about the impact of on natural communities of Cytophaga–Flavobacteria. the Cytophaga-like bacteria are to but the of this in is is about the impact of a that was recently by and The impact of can be by following the abundance of bacterial groups in with and The so far have been with freshwater assemblages. In these the abundance of or β-proteobacteria increased of but in all the and relative abundance of Cytophaga–Flavobacteria increased more so than observed in the data that Cytophaga–Flavobacteria less one of the proteobacterial subdivisions are by This however, simply from the high of these bacterial with Cytophaga–Flavobacteria will in be on more than bacteria However, that of One such is with cells be on more than There is information about for the data and of Cytophaga–Flavobacteria and other uncultured bacteria in have an the bacterial large to be by In of bacteria can when a is and thus being The to these seems to be common among the bacterial groups to and the of both proteobacteria and Cytophaga–Flavobacteria can to in other than also In heterotrophic often on some bacterial species better than on and are often higher on than on bacteria does not explain these know about vary among the major bacterial This review has the of the entire Cytophaga–Flavobacteria cluster and of three subdivisions of proteobacteria, as if these bacterial groups were of a few microbes, which of they are In we have but to use these phylogenetic because of in our of bacterial diversity in aquatic environments. in our methods new FISH for examining aquatic In we still know about the diversity of Cytophaga–Flavobacteria in aquatic habitats and about which within this cluster we should In of these however, I that the studies were able to make about the of these In of among the aquatic habitats we can see some in Cytophaga–Flavobacteria and other bacterial groups to and vary with and the in other we need more data about within the Cytophaga–Flavobacteria cluster. The what phylogenetic is most for examining the biogeochemical of Cytophaga–Flavobacteria and other uncultured bacterial The will on the specific one we know that a is needed to examine or by bacteria (but see the other it be to all heterotrophic bacteria together as a single when examining and in aquatic habitats. An against all heterotrophic bacteria together is that we now know that uncultured Cytophaga–Flavobacteria and other bacterial groups in their use of into studies of use by species (or phylogenetic which number in the to in lakes and the oceans, it be to in that current of the carbon have a single for all heterotrophic bacteria (and using even four groups (the Cytophaga–Flavobacteria and three proteobacterial would (and current Although microbial ecologists should not be by in that information about microbial community structure will be in examining larger such as the role of microbes in carbon cycles and the between community structure and is likely to our of not only microbial processes but also of the diversity of microbial communities in natural environments. are still in the of examining but even now it seems that are important members of the bacterial community in many aquatic environments. I and two for their on this review and and Glöckner for their This review was with by from the of and the