Christine Dunnington Fenton (1994)
Dept of Microbiology and Genetics, Massey
University, Palmerston North, NEW ZEALAND
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
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.
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,
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.
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
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.
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
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
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.
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
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
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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