Christine Dunnington Fenton (1994)

Dept of Microbiology and Genetics, Massey University, Palmerston North, NEW ZEALAND

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ABSTRACT

This study reports the isolation of 22 strains of Caulobacter from a variety of local water supplies. Most of the strains (17) were from the sewage treatment plant, while others were isolated from rivers (2), tap water (1) and stored water (2). Conjugative plasmid transfer was demonstrated between a strain of E. coli and a sewage Caulobacter strain. Eckhardt gel analysis and antibiotic sensitivity tests confirmed that the transconjugant Caulobacter carried a plasmid conferring neomycin resistance when compared to the neomycin sensitive parent. Caulobacter isolated from sewage tended to carry more plasmids than freshwater Caulobacter, and showed an increase in resistance to many second generation antibiotics when compared to their freshwater counterparts. Based on the sequence of a 260 bp fragment of 16S rDNA, the identities of the Caulobacter isolates were confirmed.

A phylogenetic tree constructed from the sequence data showed that the Caulobacter isolates form a diverse group. Some of the isolates appear to be closely related to marine Caulobacter and were able to grow in media containing 2.5% salt. Other isolates appear to be closely related to Pseudomonas diminuta. A number of new Caulobacter strains were identifed on the basis of their 16S rDNA sequences.

The role of Caulobacter in the environment has not been well studied, partly due to the difficulties in detecting their presence. The use of the polymerase chain reaction to amplify the 16S rDNA sequence may help to overcome this problem, bearing in mind the diverse nature of the Caulobacter group.

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INTRODUCTION

1. Discovery.

Caulobacter are stalked aquatic bacteria that are scavengers in nature. They were first discovered in 1935 after direct microscopic examination of glass slides that had been submerged in a lake for some time (Henrici and Johnson, 1935). Stalked bacteria were found adhered to the slides by virtue of an adhesive holdfast on the base of the stalk. It was not until the 1950's that Caulobacter were again noticed; this time in the water used to prepare electron microscope specimens. It was some time later in the 1960's that Caulobacter were actually isolated and maintained in pure culture (Poindexter, 1964).

2. Cell Structure.

Caulobacter are Gram negative polarly flagellate bacteria which physiologically resemble the aerobic chemoheterotrophic pseudomonads. (Poindexter, 1964) Caulobacter is unusual because cell division results in two different cell types, a stalked cell and a swarmer cell. The stalked cell is a mature cell which immediately starts replicating its chromosome in preparation for the next cell division. However, the motile swarmer cell is an immature cell which is incapable of DNA replication. In order to divide, it must differentiate by losing its flagellum and synthesising a stalk in its place. The resulting stalked cell then initiates DNA replication.

C. crescentus provides an excellent model system for studies of the temporal control of gene expression (Ely et al., 1990). Caulobacter is one of the many genera (Gram negative and Gram positive) that elaborate a paracrystalline array surface (S) layer on their outermost surface. S layers are nearly always composed of a single protein type. For most genera the function of these layers is unknown, but a protective barrier function is often presumed (Walker et al., 1992). S layer proteins share a number of physical features including a low isoelectric point pH, absence of cysteine residues, and a high proportion of hydroxy-amino acids. In several studies it has been possible to assemble the protein in the absence of the cell surface from which it was derived (Koval and Murray, 1984).

Given such similarities or capabilities, it has been suggested that some S layers were acquired by genetic exchange with other soil and aquatic bacteria and are retained because they offer a competitive advantage, analogous to antibiotic resistance or heavy metal detoxification (Walker et al., 1992). Freshwater Caulobacter are common inhabitants of aquatic and soil environments. Most isolates have S layers that are hexagonally packed and indistinguishable from each other by gross analysis. Typical strains (by laboratory analysis) have crescent shaped cells, and short stalks. Few rosettes are produced in culture but an elaborate hexagonal S layer is formed (Walker et al., 1992). Atypical strains have a variety of rod shapes; thin, straight, fat, short or long. They have larger rosettes, longer stalks and no visible S layer.

In natural environments, enrichment cultures, and pure cultures in diluted media (not more than 0.05% organic material) the length of the prosthecae or stalk exceeds the cell length by 5 - 40 times (Poindexter, 1981b). It is the ability to produce stalks coupled with the fact that Caulobacter can survive in oligotrophic environments that forms the basis of the methods for the isolation of Caulobacter. In richer media (at least 0.2% organic material) the stalk typically is much shorter. Direct microscopic examination of environments with high organic content failed to detect Caulobacter and so it was assumed that they were not present. Also, sampling of water systems usually involves the use of saline solutions and freshwater Caulobacter do not grow in salinities greater than 50 to 100 mM.

3. Distribution and Ecology.

Stalked and budding bacteria are widespread in natural ecosystems; in fresh and sea water as well as soil. These groups of bacteria may represent up to one third of the total microbial biomass (Nikitin et al., 1990). Because Caulobacter adhere to surfaces and are found in diverse locales, their role in oligotrophic environments and bacterial biofilm communities is of interest. It has been generally assumed that Caulobacter are found only in environments of low organic content but they have been enriched and isolated from a variety of sewage treatment systems (MacRae and Smit, 1991).

The sewage strains were relatively homogenous and could be reliably detected by gene probes derived from C. crescentus, a freshwater type. Most of the isolates from sewage contained one or more high molecular weight plasmids and were resistant to a number of antibiotics, characteristics not normally shared with Caulobacter isolated from other sources.

Caulobacter could be detected from virtually every type of municipal waste water treatment plant from across the USA and Canada at all points in the process except for the strongly anaerobic regions of sludge digesters used by many facilities to reduce sludge volume and generate methane gas. A recent development in waste water treatment is the 'biological' removal of phosphate from effluent. Phosphate is a key nutrient causing eutrophication of water sources as a result of sewage discharge. The process involves the accumulation of phosphate into the bacterial population as polyphosphate (Yeoman, et al., 1986). Whether Caulobacter are active participants in the phosphate accumulation process is being investigated (MacRae and Smit, 1991). Strains isolated from sewage were morphologically similar to freshwater strains. The cell bodies were crescent shaped, produced few rosettes (fused holdfasts of multiple cells) and had hexagonally packed paracrystalline surfaces (see section on Cell Structure). These isolates had increased resistance to some antibiotics such as chloramphenicol, tetracycline, erythromycin, and tobomycin. Some of these antibiotics are in common clinical use, others are 'second generation' antibiotics. These resistances may be due to plasmid transfer between antibiotic resistant intestinal or human associated bacteria and Caulobacter in the waste water treatment systems.

Freshwater Caulobacter generally had no plasmids but conjugation experiments between E. coli and freshwater Caulobacter isolates have demonstrated that antibiotic resistance transfer to Caulobacter is possible in the laboratory (Ely, 1979). Plasmid transfer between marine, freshwater Caulobacter and E.coli have also been accomplished (Ely, 1979; Anast and Smit, 1988). Because of the ability of Caulobacter to survive in oligotrophic environments, the transfer of antibiotic plasmids from coliforms to Caulobacter could aid the persistence of these plasmids in the gene pool. The significance of these observations is that Caulobacter may serve as a reservoir of antibiotic resistance determinants which then persist in the environment and be transferred back to human associated bacteria. One consequence might be a reduced lifetime for antibiotics used in clinical medicine.

Some freshwater strains appear capable of survival in a marine environment. In areas where there is storm or sewer runoff into the sea, some marine Caulobacter isolates have features which are commonly associated with freshwater strains but are rare in marine strains (Anast and Smit, 1988). One of the more diverse environments where Caulobacter have been found, apart from the gut of a millipede (Poindexter, 1964), was on unfertilised cod eggs where a long stalk was demonstrated (Hanseng and Olfasen, 1989). However, on fertilised eggs in hatching units the short stalks were more common. Reports indicate that stalked and budding bacteria were relatively abundant in intensive marine rearing units. The occurrence of Caulobacter on eggs dissected from the ovary indicated that eggs were colonised by bacteria before spawning but it is not known if this results from a pre-spawning invasion or represents an indigenous population in the Cod.

4. Oligotrophy.

An oligotrophic environment characteristically has a flux of nutrients at 0.1 mg of carbon/litre per day (Poindexter, 1981b). Most bacteria require a nutrient flux at least 50 fold higher than this. The fact that Caulobacter can survive in low nutrient environments is well established (Poindexter, 1981a). The cell can adhere to a solid surface by virtue of the adhesive material (holdfast) on the end of the stalk, allowing it to take full advantage of any nutrients which may pass by. This ability to survive in famine conditions forms the basis for the isolation of Caulobacter from the environment.

In media containing low amounts of organic material (ie. 0.01% peptone water), the bulk of 'contaminating' bacteria fail to thrive, so Caulobacter eventually become the dominant population. Coupled to this, the stalk elongates in low phosphate conditions which is in itself the main diagnostic feature for the detection and isolation of Caulobacter. It is known that in phosphate sufficient environments some Caulobacter strains do not produce the long stalks that are characteristic of the genus in phosphate limited situations, and so can be difficult to identify by light microscopy.

The concentration of at least one inorganic nutrient, phosphate, is inversely proportional to the length of the appendage (stalk), a relationship seen in other prosthecate bacteria (Poindexter, 1981b). Accordingly stalk elongation is regarded as a morphological response to nutrient limitation and can be interpreted as a means of increasing the surface:volume ratio of the cell in dilute environments. A stalked cell whose appendage is ten times the cell length has a surface:volume ratio that is twice that of the cell alone. Even more important with respect to increasing the ratio of potential uptake sites to metabolically active cytoplasm, the Caulobacter appendages are composed almost entirely of membranes, which are generally inactive as sites of energy consuming biosynthesis and lack complete catabolic systems (Poindexter, 1981b). The cross walls peculiar to Caulobacter prosthecae may serve to restrict the entry of the cytoplasm into the stalk so that its contribution as an uptake organelle is not reduced by substrate consuming reactions.

Caulobacter are able to accumulate poly-b-hydroxybutyrate (PHB) and polyphosphate and can sometimes grow in anaerobic conditions. Under conditions of nitrogen or phosphate limitation, 26% of the dry cell weight can be attributed to PHB (Poindexter, 1981b). Cells provided with glucose but without a nitrogen source increased in dry weight by 21% in 12 hrs with 90% of the increase being accounted for by the synthesis of PHB and of poly-glucose (Poindexter, 1981b). Earlier cytological studies revealed that under conditions of nitrogen starvation in a sugar phosphate medium, the cells also accumulated polyphosphate reserve granules (Poindexter, 1981b).

It is concluded that Caulobacter has the capacity to form all three principal types of reserve polymers simultaneously and are able to survive during periods of nutrient exhaustion.

5. Taxonomy.

In the case of Caulobacter, what morphologically appears to be a Caulobacter will generally be called one without challenge. This is mainly due to a lack of other defining physiological or metabolic traits (Stahl et al., 1992). The Caulobacter group has been well studied and in the past the taxonomy of this group has been based on morphological criteria and required growth factors (Poindexter, 1989). 16S rRNA analysis has shown members of Caulobacter to be members of the alpha subdivision of Proteobacteria ( Stackebrandt et al., 1988). This group includes non-phototrophic and non-budding organisms (Albrecht et al.,1987).

The budding and/or prosthecate non-phototrophic bacteria include the genera: Hyphomicrobium, Hyphomonas, Pedomicrobium, Filomicrobium, Stella and Caulobacter. Three large groups can be distinguished among this group: caulobacter-like, hyphomonas-like and hyphomicrobium-like bacteria (Nikitin et al., 1990). Relatively little information is available concerning the genetic diversity of prosthecate bacteria. Early DNA hybridisation (Moore et al.,1978) and more recent 5S and 16S rDNA sequence comparisons (Lee and Fuhrman, 1980; Nikitin et al., 1990; and Stackebrandt et al.,1988) suggest that there is considerable diversity among this group. 16S rDNA analysis by comparative sequencing of 'typical' Caulobacter strains found them to be a relatively closely related subgroup of freshwater isolates while atypical strains were different from the typical cluster and from each other (Stahl et al., 1992). Typical Caulobacter were still measurably dissimilar exhibiting rRNA similarity values of about 99% (DNA similarities of 50% generally correspond to rRNA similarity values of 98 to 99%, Stahl et al., 1992).

The most distantly related of the Caulobacter characterised were associated at approximately 88% 16S rDNA sequence similarity. Notably affiliation with either one of the two phylogenetically distinct lines of descent (88 to 90% similarity) generally corresponded to a marine or a freshwater habitat. One line of descent was composed exclusively of marine Caulobacter. The other line of descent included the freshwater Caulobacter and some marine isolates. Most Caulobacter isolated from waste water treatment systems belonged with the terrestrial or freshwater lineage (Stahl et al., 1992). An apparent exception to this pattern was of C. subvibrioides which morphologically would be included in the genus Caulobacter but is phylogenetically distinct from both the terrestrial and the marine types (Stahl et al., 1992). The cloned paracrystalline surface (S) layer gene of C. crescentus CB15A hybridised to specific regions of the genome for most of the Caulobacter analysed under moderate stringency conditions (Walker et al., 1992).

Restriction fragment length polymorphism analysis with the S layer gene as the probe, failed to reveal patterns of close relatedness between the strains. This indicates a greater genetic diversity than is suggested by morphological similarities. This correlates with 16S rDNA comparative analysis that showed that these Caulobacter were a coherent group but still sufficiently different to have significant variation in their overall genomic DNA composition. When a flagella filament protein gene was used to probe a group of non-Caulobacter isolates from waste water treatment systems, one strain in 150 isolates hybridized with the probe DNA (MacRae and Smit, 1991). This isolate was examined by the Biolog commercial identification scheme (which does not include Caulobacter) and a match to Pseudomonas vesicularis was obtained (Stahl et at., 1992). This species is similar to P. diminuta on the basis of RNA homology and these two species form a highly distinctive branch of pseudomonads (Gilardi, 1985). Also, one of the freshwater Caulobacter when examined by the Biolog system, scored an acceptable match to P. diminuta.

It is conceivable that these species are Caulobacter strains locked in the motile phase. By classical definition, a bacterium which does not posses a stalk, cannot be called a Caulobacter. A stalk-less Caulobacter might be identified as a pseudomonad since they are physiologically similar. A comparison of rDNA gene sequences is needed to confirm the relationship between Caulobacter and Pseudomonas diminuta.

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DISCUSSION

1. Isolation and Enrichment

There are many publications on the genetics of Caulobacter, mainly because of the dimorphic life cycle, but very little on the microbiology and ecology of it. Most of the studies were carried out on a few environmental isolates, some of which were isolated as early as the 1960’s (Poindexter, 1964) and have been in laboratory culture ever since.

1.1 Identification of Caulobacter in Enrichment Cultures

The literature which dealt with the enrichment and isolation of Caulobacter (Poindexter, 1964; Scmid, 1981; MacRae and Smit, 1991) failed to deal adequately with the problems associated with the isolation of Caulobacter from an enrichment culture. Most of the publications had photographs of isolates in a purified form which does not always represent the morphology of a Caulobacter in an enrichment culture. The length of stalk, the formation of rosettes and the cell shape can appear different. Photographic evidence of the appearance of Caulobacter cells in an enrichment culture (as in this thesis) would have been useful. During the course of this investigation, it was found that a wet mount was preferable to staining for the detection of Caulobacter cells. Caulobacter cells which had long stalks, as was usually the case, were detectable by their swaying movement. Focusing at different depths of field near the area of movement usually revealed stalked bacterium.

1.2 Problems with the isolation of Caulobacter

The conditions under which the enrichment culture is incubated can influence the type of Caulobacter strains that dominate the population. The type of population present is influenced by the amount of illumination that the culture has, the amount of algae which is present, and the time of year that the sample was taken (Schmid, 1981). Most of the strains mentioned in publications were isolated from Northern America. Based on information taken from the literature, it was decided that a pigmented Caulobacter would be the most common type present in the enrichments, under the conditions used in this investigation. Only one of the strains from sewage was pigmented and the isolation of non-pigmented strains took longer than expected as they were initially over-looked. This is the first reported isolation of New Zealand Caulobacter species.

One of the enrichment cultures (Tiritea stream) contained a lot of another type of prosthecate bacteria (Hyphomicrobium, plates 6, 7 and 8). According to the literature reviewed, the media (PYEA, section 1.2.1) and procedure used to isolate Caulobacter strains should not have been suitable for the isolation of Hyphomicrobium (Poindexter, 1989). However, every initial attempt at the isolation of Caulobacter from the Tiritea stream resulted in the isolation of Hyphomicrobium. The water from Taranaki Base Hospital was the only sample taken where Caulobacter was not isolated. The hospital had been having a series of problems with contaminated water at the Blood Bank Unit. The contaminant appeared to be a "webbed" bacteria (Dean Anderson, personal communication). The New Zealand Centre for Disease Control (Porirua, New Zealand), identified the contaminant as Pseudomonas fluorescens. As strains of Caulobacter and some Pseudomonas species have been shown to be closely related, it was considered that the contaminant might have been a mis-identified Caulobacter. Caulobacter were present in the enrichment but not in sufficient numbers for it to be successfully isolated, nor to conclude that they were the mass contaminant.

In general, the best way to isolate Caulobacter is by the surface film method (Materials and Methods, section 1.7.) using PYE medium and a long incubatiuon period. The majority of the strains used in this study were isolated using this method but with a modification to published procedures in Poindexter, (1964) and MacRae and Smit, (1991). The surface film samples were washed repeatedly in 0.1% sarcosine to disperse clumps of bacteria and separate the cells before they were streaked onto solid media. The length of time taken to isolate Caulobacter can sometimes be shortened by using the attachment and the physical isolation methods (Materials and Methods, section 1.6.1, 1.7.2) in conjunction with low phosphate PYE or PCa medium (Materials and Methods, section 1.2.2, 1.2.5). The low phosphate PYE medium helps in the detection of Caulobacter on solid media, because the stalks are elongated under low phosphate conditions (Poindexter, 1981b). For some Caulobacter isolates, the presence of yeast extract in the culturing medium can inhibit prosthecate development (Poindexter, 1989). PCa medium has no yeast extract. However, unless an enrichment culture has a high yield of stalked bacteria in the surface film, isolation is still difficult. None of the literature examined addressed the difficulties with purifying a bacterium that can adhere to other bacteria or debris. Normal streak plating methods often failed to completely disperse the Caulobacter cells even after they had been washed in 0.1% sarcosine and vortexed. As a final purity check, each isolate was grown in PYE broth (inoculated from a single colony) and 0.1 ml was spread on solid media as outlined in Materials and Methods section 1.8. Colonies that had arisen from contaminating cells were obvious in the lawn.

(TO BE COMPLETED......)

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(Reproduced with permission)