Elongation Correlates with Nutrient Deprivation in Pseudomonas aeruginosa Unsaturated Biofilms

Abstract

Bacteria in nature frequently grow as biofilms, yet little is known regarding how biofilm bacteria morphologically adapt to low nutrient availability, which is common in unsaturated environments such as the terrestrial subsurface or on plant leaves. For unsaturated biofilms, in which the substratum may provide all nutrients, what are the relationships between nutrition and cell size and shape-the simplest metrics of cellular morphology? To address this question, we cultured Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium that is environmentally and medically important, on membranes overlaying solid media, and then measured cellular dimensions using atomic force microscopy (AFM). Nutrition was controlled chemically by media composition and physically by stacking membranes to increase the path length for nutrient diffusion. Under conditions of carbon-nitrogen imbalance, low carbon bioavailability, or increased nutrient diffusional path length, cells elongated while maintaining constant width. A mathematical relationship suggests that, by elongating, biofilm bacteria strategically enlarge their nutrient collection surface without substantially changing the ratio of surface area to volume (SA/V). We conclude that P. aeruginosa growing as unsaturated biofilm with a planar nutrient source morphologically adapt to starvation by elongating. This adaptation, if generalizable, differs from a better-understood starvation response (i.e., cell size decreases; thus SA/V in-creases) for planktonic bacteria in well-mixed environments.

The Selective Value of Bacterial Shape

“…When limited for biotin, Arthrobacter globiformis forms abnormally large, branched rods of variable size (365), as do other isolates when starved for manganese (56,89). An analogous magnesium deficiency inhibits cell division and produces nonbranching filamentation in Clostridium welchii (355,356), and in nutrientpoor conditions Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens elongate into long slim cells, unlike the short rods observed in liquid medium (302,314). The simplest explanation for these responses is that, when the environment demands it, many bacteria can accelerate or delay cell division and septation, thereby creating shorter or longer cells, respectively.…”

“…This may be reason enough for cells in suspension. Second, filamentation may benefit cells attached to a surface, not because elongation increases the total surface area but because it increases that specific surface (314). For a rod whose length is 7 times the sphere's diameter the contact surface increases to 23%, but a rod 10 times as long increases its contact area to only ϳ24%, and further elongation has little additional effect (314).…”

“…Second, filamentation may benefit cells attached to a surface, not because elongation increases the total surface area but because it increases that specific surface (314). For a rod whose length is 7 times the sphere's diameter the contact surface increases to 23%, but a rod 10 times as long increases its contact area to only ϳ24%, and further elongation has little additional effect (314). Thus, a rod seven times as long as a coccus increases its surface contact by ϳ40%, which should be sufficient to favor rod-shaped cells if surface contact is the principal source of nutrients.…”

Biofilm Cohesiveness Measurement Using a Novel Atomic Force Microscopy Methodology

“…Cell surface hydrophobicity and charge have also been investigated using chemically functionalized AFM probes (2, 41). All of these studies and measurements provide important information on single-cell properties; nevertheless, they do not provide information on the properties of whole biofilms.Because of the difficulties associated with working with biofilms, particularly their softness and gelatinous nature, most biofilms imaged by AFM have been dried first (8,25,35). Drying is expected to significantly change the strength and…”

“…Because of the difficulties associated with working with biofilms, particularly their softness and gelatinous nature, most biofilms imaged by AFM have been dried first (8,25,35). Drying is expected to significantly change the strength and…”