papers

Prosthecobacter fusiformis nov. gen. et sp., the fusiform caulobacter

summery

they explain their findings and justify why the organism should be classified as a new species.

• they collected samples from freshwater sources.
• four strains of heterotrophic fusiform caulobacters were isolated from these samples.
• morphologically, these bacteria resemble Caulobacter. they possess a single prostheca extending from one pole of mature cells, which aids in attachment to various substrates through a holdfast located at the distal tip of the appendage.
• however, they differ in several key aspects:
  • they lack the dimorphic life cycle of Caulobacter. Instead of producing motile, non-stalked daughter cells, their daughter cells are immotile and are identical mirror-image replicas of the mother cells.
  • the prosthecae of these fusiform caulobacters lack crossbands, are wider, and end in a bulbous tip.
  • their DNA base composition (GC content) ranges from 54.6% to 60%, whereas Caulobacter has a GC content of 62–67%.

notice that bacteria are living a 3D life and not a 2D view you see under a microscope.

introduction

they outlined the historical progression of discoveries related to this strain, from the first scientist who identified it to the present findings.

• in 1935, henrici and johnson noted that stalked bacteria are an important component of the bacterial periphyton in lakes. they observed unicellular organisms with a single polar stalk and proposed the genus caulobacter, distinguishing two morphologically distinct types: one, a vibrioid to rod-shaped stalked cell that produced non-stalked daughter cells (possibly motile), and the other, a fusiform type, which they inferred to be immotile. the fusiform type’s daughter cells attached to the slides, but they were unable to isolate them.

• in 1951, houwink developed a method for isolating this genus. he discovered that the stalk was an outgrowth of the cell rather than an excreted appendage, leading to the term prostheca (staley, 1968).

• in 1962, stove and stanier observed caulobacter and noted that mother cells gave rise to polarly flagellated, non-prosthecate daughter cells (swarmer cells), which later lost motility and developed a stalk in place of the flagellum.

• in 1964, pondexter discovered asticacaulis, which had subpolar appendages. similar to caulobacter, it exhibited a dimorphic life cycle, where a flagellum was later replaced by a prostheca. typically, asticacaulis had biprosthecate cells.

• in 1970, we isolated a non-motile fusiform caulobacter fitting the description of henrici and johnson. initially, we obtained one isolate (which was not reported), followed by three more, and now we present our findings.

materials and methods

individual colonies were examined in phase wet mounts to locate colonies of these organisms.

translation

• Colonies were prepared using a wet mount technique.
• They were viewed under a phase-contrast microscope.
• This tool enhances visualization of transparent or unstained organisms.

temperature range for growth was tested in a gradient block (Matches and Liston, 1973) over the range from 0 to 40C after incubation for 30 days.

translation

• Temperature Range:
  • The experiment aimed to determine the temperatures at which a microorganism can grow.
  • The range tested was from 0°C to 40°C.
• Gradient Block:
  • A gradient block is a device or method used to expose microorganisms to a range of temperatures simultaneously.
  • This setup allows researchers to test growth conditions efficiently over a continuous temperature spectrum.
• Incubation Period:
  • The test duration was 30 days, ensuring sufficient time for slow-growing organisms to exhibit growth.
• Reference:
  • The study referenced is by Matches and Liston (1973), likely indicating that the gradient block method used was based on their work.

why use phase microscopy? (When to use it)

• Phase contrast microscopy is particularly useful in this scenario because it:
  • Allows observation of live cells without staining, preserving their natural morphology.
  • Highlights differences in refractive index, making even subtle contamination visible.

• strains. strains needed to be isolated and cultivated using standard methods to ensure reproducibility. they followed houwink’s protocol, which employs a 0.01% peptone culture—a minimal nutrient medium designed to prevent the overgrowth of organisms that thrive in richer media.
  • enrichment cultures (with 0.01% peptone) were incubated for 1–3 weeks, allowing bacterial populations to reach appreciable numbers.
  • 0.01% peptone agar plates were prepared to transfer the cultures for colony isolation.
  • agar plates were incubated for 1–2 weeks at room temperature.
  • individual colonies were examined using phase-contrast microscopy on wet mounts.
  • samples were collected from raw sewage, a wastewater treatment facility, a university lake, a pond near another university, and a marl pond.
• media. various culture tubes were prepared to assess carbon source utilization and vitamin requirements.
  • vitamin requirements were tested using a defined medium, with three consecutive transfers in DM (defined medium) with and without the vitamin solution.
  • carbon source utilization was assessed using DM without glucose, with each compound added at a final concentration of 0.2% (w/v) after sterilization and filtration.
  • cultures were incubated for 30 days at 30°c.
  • phase-contrast microscopy was used to determine cultural purity.
  • for all other experiments, a complex medium (MMB) was used, distinguishing these cultures from the enrichment cultures.
• physiological tests.
  • temperature range tolerance was tested using a gradient block (0–40°c) after 30 days of incubation.
  • anaerobic growth was tested in MMB with and without alternative electron acceptors, following incubation in a gaspak container.
  • dna was extracted using the marmur method, and base composition was determined by buoyant density centrifugation in CsCl.

• electron microscopy. cells were stained with 0.2% PTA and other contrast agents.

• life cycle studies. photomicrographs were taken at periodic intervals using a zeiss ultraphot microscope.

• phage typing. phages were obtained from various institutes, and plates were incubated for one week.

results

they also grew in a defined medium in which ammonium served as the sole nitrogen source providing an appropriate carbon source was present.

translation

• "Providing": Used to set a condition (if).
• "An appropriate carbon source": A source of carbon that the organism can utilize for growth.
• "Was present": Ensures that this source is physically available in the medium.

morphology by light microscopy.

• they are unicellular.
• shape of fusiform rods to fusiform vibrios.

• FC4
  • fusiform rod, some cells were slightly bent.
  • length: before division 8-10μm, after division 3μm.
  • diameter: 0.5μm.
• FC2
  • resembles FC4.
• FC3
  • definite vibrioid cells.
  • length: 2μm to 8μm. (shorter than others)
  • width: same as FC2 and FC4.
• FC1
  • width: 1μm (wider than others)
• rosettes rarely found with few cells.
• motility has not been observed in any strains.

appearance by electron microscopy.

• This statement means that the general shape of the bacteria observed under phase contrast microscopy (a type of light microscopy) was validated and confirmed by using electron microscopy.
• all strains had fimbrae coating the entire surface except for the distal end of the prostheca.

• fully differentiated prostheca’s width: 0.1-0.2μm (other caulobacters’ : 0.085μm.)
• they have a characteristic bulbous tip. (it means it is distinctive and defining feature.)

• they completely lack crossband structures (querbalken).
• there was some variation in the extent to which cells had differentiated prior to separation.

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fine structure.

• cytoplasmic constituents including ribosomes were found extending into the prostheca of FC3.
• cell wall, gram negative.

growth cycle.

• the cell elongates at the nonprosthecate pole.
• cell septum forms and the daughter cell differentiates.
• newly formed prostheca was usually shorter than that of the mother cell at the time of cell seperation.

cultivation and physiology.

• chemoheterotrophic (derives its energy from the oxidation of chemical compounds and obtains its carbon from organic sources.
• growing well in complex medium with sugar as carbon source.
• also grow well in a medium in which ammonium served as the sole nitrogen source.
• carbon sources used: glucose, galactose, mannose, sucrose, lactose, melibiose, maltose, turanose.
• they didn’t use sugar alcohols (sorbitol, mannitol, arabitol, erythritol) or alcohols or 20L-and DL-amino acids.
• didn’t need vitamins for growth.
• all were mesophiles:
  • FC1: 1-35 °c
  • FC2: 10-38 °c
  • FC3: 8-38 °c
  • FC4: 4-40 °c
• obligate aerobes. (incapable of growing anaerobically)
• catalase-positive.
• oxidase-negative.
• forming convex colonies with entire margins in agar plates.
• FC4 and FC3 were opaque light yellow, FC2 and FC1 were opaque white.
• dna base composition:
  • FC1: 54.6
  • FC2: 60.1
  • FC3: 57.1
  • FC4: 56.1
• none of Caulobacter and Asticcacaulis phages formed plaques on any of them.

discussion

• superficially the fusiform caulobacters described herein resemble Caulobacter.
  • similarities:
    • they have a single polar appendage (to attach substrata with holdfest at the tip of it)
    • they grow in dilute organic media, sharing certain nutritional and physiological features with Caulobacter and Asticcacaulis.
  • differences:
    • prosthecobacteria don’t have a dimorphic life cycle with non-motile, prosthecate mother cells and motile non-appendaged daughter cells.
    • mother cell divides to give rise to a daughter cell which is an identical mirror image of the mother cell at the time of seperation.
    • the prostheca is wider and terminates in bulbous tip in fully differenciated cells.
    • no crossbands.
    • no flagella, fimbrae are numerous.
    • phages of Calobacter and Asticcacauli can’t affect them.
    • dna base composition value: 54.6 to 60.1/
• then they propose it as a new genus and discuss naming.

the original paper