Methylotrophic growth on MSA.

Bacteria have been shown to use the phosphate analogue of MSA, methane phosphonate (MPA). This is degraded by bacteria such as Pseudomonas testosteroni and Escherichia coli, with the release of methane (Daughten et al, 1979a, 1979b; Cook et al., 1979; Ghisalba et al., 1986).

No organism has been isolated with the capability to derive carbon and energy from MSA, despite the entry of the

compound to the natural environment for at least ten thousand years (Saigne and Legrand, 1987). It seemed unreasonable to suppose that this stable organic acid

persisted in the soil. Indeed, alkane sulphonates are known to be biodegradable. Pseudomonas species were readily

isolated from garden soil and surface water with alkane sulphonates (C4-C12) as their only source of carbon and energy, releasing an aldehyde and sulphite (Thysse and Wanders, 1972, 1974).

In enrichment culture, MSA could have been considered a carbon source suitable for methylotrophic growth, with oxidation of the methyl group providing energy.

Chemolithotrophic growth, with cleavage of the C-S bond and the oxidation of released sulphite providing energy, would also have been possible. Thus an enrichment culture could have yielded restricted methylotrophs, facultative

methylotrophs or Thiobacillus-type organisms. Another possibility was that MSA could only be used as a sulphur source.

Biogeochemical data suggested that MSA was present

throughout the environment (See section 1.4), and so an enrichment from garden soil was chosen.

6.2 Enrichment and isolation of methane sulphonate users

The low phosphate medium (MinE) chosen for the enrichment of bacteria capable of using MSA as sole carbon and energy source was that of Owens and Keddie (1969). Originally developed for the isolation of coryneform bacteria, it was adapted by L.J. Zatman (University of Reading) in later years to serve as a medium for the isolation of

methazotrophs (i.e. those bacteria using MMA as sole carbon, energy and nitrogen source). Since MinE supported the growth of a wide variety of facultative methylotrophs as well as more conventional heterotrophs, it was once again adapted, to maximise the recovery of MSA-users.

In order to introduce some degree of selectivity in the properties of the medium, sulphur-containing compounds, such as magnesium sulphate, were omitted (see composition of MinE and MinE-S in Sections 2.2.6.1 and 2.2.6.2 respectively). These were replaced by the chloride form of the compound, in an effort to maintain the ionic balance of the medium. This policy was not extended to the trace metal or vitamin

solutions, which had a very low concentration of sulphate. It was hoped that by limiting the sulphur available,

conditions would favour those organisms capable of using MSA

as sulphur and/or carbon source. A measure of the effectiveness of this approach can be gained from the observation that conventional methylotrophs, such as

Bacillus PM6, would not grow in the adapted medium even in the presence of MMA, that organism's isolation substrate.

Attempts by second year undergraduates (at the University of Warwick) to isolate MSA-users using NMS (see Chapter 2, section 2.2.5.3) failed. Similarly, the number of MSA-users isolated from MinE enrichment and purification culture was less than that from MinE-S (Murrell, J.C. and others, University of Warwick, unpublished results.). This would seem to indicate that the choice of MinE and the omission of sulphur provided an almost ideal medium for the isolation of MSA-users.

Inoculating soil and water samples into media containing MSA as sole carbon and energy source did not give rise to

organisms able to grow on MSA. Thus it was decided to use a chemostat-enrichment procedure with both methylamine

hydrochloride (MMA) and MSA. This would have enhanced recovery of methylotrophic bacteria that might also have used MSA as a sole or co-substrate, and would have also allowed enrichment of specialist MSA-users unable to use MMA, assuming that these organisms could have competed

successfully with other organisms. Such procedures have been successful in isolation and competition experiments with sulphur-using bacteria (Smith and Kelly, 1979; Kelly and Kuenen, 1984)

The decision to add MMA was also influenced by the

possibility that MSA could only act as a sulphur source for bacteria (see above). If this was indeed the case, then methylotrophs using MSA as sole sulphur source would have been isolated.

Since no bacteria had previously been isolated using MSA, the potential toxicity of the sulphonate was unknown. In preparing media for the enrichment, the concentration of carbon as MMA and MSA was limited to 10 mM (7 mM MSA + 3 mM MMA), as this was a carbon source concentration used for the growth of known methylotrophs.

The chemostat was inoculated with 100 g of garden soil, taken from under a rose bush. The proximity to a rhizosphere would have encouraged a diversity of organisms to grow or survive in the soil. The chemostat equipment was used as a batch culture for the first few weeks. After this time fresh medium was fed in for one hour in every three and then

continuously. Five weeks after inoculation, the enrichment was visibly turbid and it was decided to take a sample and serially dilute it, plating the dilutions onto various agars

(nutrient agar, MinE + 10 mM MMA and MinE + 10 mM MSA). Organisms showing seven different colony forms were

subcultured from these plates. Nine cultures were obtained from these, growing on MSA as sole carbon and energy source. They were named M1-M9.

6.3 Characterisation of Strains N1-H9

6.3.1 Morphology

The morphologies of the bacteria isolated capable of using MSA as sole carbon and energy source are listed in table 6.3.1.1.

strain Number Morphology Gram stain Origin

Ml Rod, single — _Chemostat

M2 Rod, single/pairs - Chemostat

M3 Rod, single + Chemostat

M4 Rod, single - Chemostat

M5 Slender rod + Chemostat

M6 Rod, single - Chemostat

M7 Rod, single - _Chemostat

M8 Coccus + Chemostat

M9 Rod, single - Chemostat

M56 Rod, single Aerial

Table 6.3.1.1 The eorphologies of the MSA users M1-M9 and H56. M56 was an aerial contaiinant isolated froi a MinE-S plate

6.3.2 Selection of M2 for further study

Although Ml and M3-M8 were purified to single colonial and morphological types, their colonies were small (<1 mm) and often difficult to see on MSA agar plates. Moreover, growth in liquid culture (with MSA as sole substrate) took 7 days or more to reach visible turbidity. However, the isolate M2 grew as white colonies, 1-2 mm in diameter, on MSA agar and produced visibly turbid MSA liquid cultures within three days. Quite evidently M2 had a far better yield on MSA than any of the other isolates and so was more suited to further biochemical and microbiological study. Furthermore, Ml and M3-M9 did not survive repeated subculture on agar and so