Nicholas J. Russell

Mechanisms of thermal adaptation in bacteria: blueprints for survival

Abstract Bacteria use a variety of mechanisms to alter the lipid composition of their membranes, and hence membrane fluidity, in response to changes in growth temperature. These modifications are an important feature of adaptation, enabling

Adaptive modifications in membranes of halotolerant and halophilic microorganisms.

Halotolerant and halophilic microorganisms can grow in (hyper)saline environments, but only halophiles specifically require salt. Genotypic and phenotypic adaptations are displayed by halophiles; the halotolerants adapt phenotypically, but it is not established whether they show genotypic adaptation. This paper reviews the various strategies of haloadaptation of membrane proteins and lipids by halotolerant and halophilic microorganisms. Moderate halophiles and halotolerants adapt their membrane lipid composition by increasing the proportion of anionic lipids, often phosphatidylglycerol and/or glycolipids, which in the moderately halophilic bacteriumVibrio costicola appears to be part of an osmoregulatory response to minimize membrane stress at high salinities. Extreme halophiles possess typical archaebacterial ether lipids, which are genotypically adapted by having additional substitutions with negatively-charged residues such as sulfate. In contrast to the lipids, it is less clear whether membrane proteins are haloadapted, although they may be more acidic; very few depend on salt for their activity.

The purification and chemical characterisation of the alginate present in extracellular material produced by mucoid strains of Pseudomonas aeruginosa

A rapid ion-exchange method has been used to purify the alginate from the extracellular material of mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients. The structure has been investigated by chemical analysis, infrared spectroscopy, paper chromatography, and gas-liquid chromatography. The alginates contain mainly random or poly(D-mannuronic acid) block structures, and are highly acetylated. The relative viscosity is not correlated with the ratio of D-mannuronic acid to L-guluronic acid residues, or the degree of acetylation. The chemical/physical properties of the alginate from P. aeruginosa are considered in the context of the growth of the organism in the lung.

The Lipid Composition of Azole-sensitive and Azole-resistant Strains of Candida albicans

The lipid compositions of two azole-sensitive (A and B2630) and two azole-resistant (AD and KB) strains of the opportunistic fungal pathogen Candida albicans were studied by using several lipid extraction procedures: no differences were observed between the lipid content or total phospholipid/neutral lipid ratios of the four strains. All contained phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylserine as major phospholipids, with smaller amounts of phosphatidylglycerol and diphosphatidylglycerol; the relative proportions of these lipids differed between all four strains. The fatty acid composition of each major phospholipid within each strain differed, and there were also interstrain differences. A marked effect of culture growth phase in batch culture on lipid composition was observed. The major neutral lipids in each strain were triacylglycerol, non-esterified sterol and non-esterified fatty acid. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids, and there were also interstrain differences. All strains possessed (lyso)phospholipase activity, which was non-specific. The proportions of triacylglycerol and non-esterified fatty acid did not vary between strains, but the azole-resistant strains AD and KB contained more non-esterified sterol, giving them a phospholipid/sterol ratio approximately half that of azole-sensitive strains. There appeared to be a relationship between the phospholipid/sterol ratio of exponentially growing sensitive strains and their ability to take up azole; this did not extend to the resistant strains, which either did not take up azole (AD and KB) or took it up at a faster rate (Darlington) than sensitive strains.

Citrate synthase and 2-methylcitrate synthase : structural, functional and evolutionary relationships

Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.

SDS-degrading bacteria attach to riverine sediment in response to the surfactant or its primary biodegradation product dodecan-1-ol

A laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonas strains to natural river-sediment surfaces. Three of the Pseudomonas strains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.

Modelling the kinetics of biodegradation of anionic surfactants by biofilm bacteria from polluted riverine sites: A comparison of five classes of surfactant at three sites

The capacity of epilithic bacteria from different riverine sites to biodegrade representatives of five classes of anionic surfactants, was quantified by analysis of biodegradation kinetics in die-away tests. The sampling sites in the River Ely, South Wales were located near a sewage treatment plant outfall. Indigenous stones from the riverbed were removed from sites immediately beneath the outfall (BO), and at sites 15 m upstream (BU) and 50 m downstream (BD). The five surfactants used were sodium dodecyl sulphate (representing primary linear alkyl sulphates, PLAS), EmpicolRESB3S (alkyl ethoxy sulphates, AES), potassium undecyl-2-sulphate (secondary linear alkyl sulphates, SLAS), sodium 1-dodecane sulphonate (primary alkane sulphonates, PAΣ) and 5-phenyl dodecane sulphonate (linear alkyl benzene sulphonates, LABΣ). Irrespective of sampling site, the order of biodegradability assessed as the reciprocal of half-time for surfactant disappearance was PLAS > AES > SLAS > PAΣ > LABΣ. Within each surfactant class, the half-time decreased in the order BU > BD > BO. Non-linear regression analysis of the die-away data to several kinetic models which combined disappearance of surfactant by zero, first order or Michaelis-Menten kinetics, with growth (exponential or logistic) of competent cells, yielded as best fit the model involving exponential growth of competent cells with first-order disappearance of substrate. This model generated two biologically-significant parameters: K1 which is equivalent to first-order rate constant in the die-away test at t = 0 (i.e. before growth distorts the population from its indigenous composition); and r, the specific growth rate of the biodegrading population in the die-away flask. For a given site, K1 decreased in the order PLAS > AES > SLAS > PAΣ > LABΣ, reflecting their relative biodegradability. Values of r for the primary sulphate esters (PLAS and AES) were always greater than values for the other surfactants, especially the sulphonates (PAΣ and LABΣ), indicating that different sub-populations existed for the biodegradation of these classes. Moreover, lack of inter-site variation in this pattern suggested similar distributions of surfactant-biodegrading populations at each site. Values of {K1viable-cell count}, which is a measure of surfactant-biodegrading activity per unit of population, fell into two groups: higher values (i.e. > 10−9h−1 cfu−1 ml) with little inter-site variation (∼3-fold) for sulphated surfactants, and lower values (mostly 30-fold) for sulphonates. These results indicated that the capacity to biodegrade sulphated surfactants was more widely distributed in bacteria than was the capacity to biodegrade sulphonates. This situation may have evolved as a result of selective pressure from exposure of bacteria to environments in which natural analogues of sulphated surfactants, but not of aryl sulphonates or LABΣ, are commonly present.

The growth and phospholipid composition of a moderately halophilic bacterium during adaptation to changes in salinity

The effect of a sudden change in NaCl concentration of the medium on the time course of alterations in growth rate and phospholipid composition of the moderately halophilic bacteriumVibrio costicola has been investigated. This organism and other moderate halophiles are known to contain a larger proportion of negatively charged phospholipids in their membranes when grown at higher salt concentrations. We show for the first time that the change in proportion of phosphatidylglycerol, relative to phosphatidylethanolamine, which occurs after a shift from 1M to 3M NaCl, or vice versa, is essentially completed during that period immediately following the salt shift when growth is zero or very slow, and before the cells have adopted the growth rate appropriate to the new salt concentration. It appears, therefore, that the alteration in membrane phospholipid composition may be a necessary physiological response for adaptation to change in salinity.

The effect of salinity and compatible solutes on the biosynthesis of cyclopropane fatty acids in Pseudomonas halosaccharolytica

SUMMARY: The moderately halophilic eubacterium Pseudomonas halosacchavolytica has been grown at salinities over the range 5-25 % (w/v), equivalent to 0.7-3.5 M-NaCl, and the fatty acid composition determined in the late-exponential and stationary phases of batch culture. There was an increase in the proportion of cyclopropane fatty acids (CFA) as the cultures went into stationary phase at all salinities; the overall proportion of CFA was higher in the media containing more salt. The biosynthesis of CFA in P. halosacchavolytica was determined using radiolabelled S-adenosylmethionine as the precursor incubated in cell-free extracts prepared by breaking bacteria with a French press. Compared with the activity obtained in 100 mM-phosphate buffer, the activity of CFA synthetase was inhibited by the addition of NaC1 or KC1, but stimulated up to 12-fold by added glycinebetaine, with maximum activity at 3 M. Although the specific activity of CFA synthetase in lysates from cultures grown in 0.7 or 2.1 M-NaC1 were similar in the presence of 3 M-glycinebetaine, the enzyme activity in low-salinity cultures was better adapted to function in 1 M-glycinebetaine. Shift-up experiments, in which CFA synthetase activity was assayed in cell-free extracts prepared at different times after increasing culture salinity from 0.7 to 2.1 M-NaC1, showed that the activity of the enzyme was immediately responsive to compatible solute concentration changes and indicated that enzyme induction would not be required to achieve the salt-dependent alterations in membrane lipid CFA composition in vivo. A range of other compatible organic solutes stimulated CFA synthetase activity to a much lesser extent (1.8-fold) compared with glycinebetaine. It is suggested that a compatible solute, which is normally accumulated during osmo(halo)adaptation by an organism in order to contribute towards osmotic balance, does not behave passively towards intracellular proteins but can also stimulate enzyme activity.

GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa : evidence from metabolite studies

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.