Leptothrix cholodnii Response to Nutrient Limitation

Abstract

Microorganisms are widely utilized for the treatment of wastewater in activated sludge systems. However, the uncontrolled growth of filamentous bacteria leads to bulking and adversely affects wastewater treatment efficiency. To clarify the nutrient requirements for filament formation, we track the growth of a filamentous bacterium, Leptothrix cholodnii SP-6 in different nutrient-limited conditions using a high aspect-ratio microfluidic chamber to follow cell-chain elongation and sheath formation. We find that limitations in Na+, K+, and Fe2+ yield no observable changes in the elongation of cell chains and sheath formation, whereas limitations of C, N, P, or vitamins lead to more pronounced changes in filament morphology; here we observe the appearance of partially empty filaments with wide intercellular gaps. We observe more dramatic differences when SP-6 cells are transferred to media lacking Mg2+ and Ca2+. Loss of Mg2+ results in cell autolysis, while removal of Ca2+ results in the catastrophic disintegration of the filaments. By simultaneously limiting both carbon and Ca2+ sources, we are able to stimulate planktonic cell generation. These findings paint a detailed picture of the ecophysiology of Leptothrix, which may lead to improved control over the unchecked growth of deleterious filamentous bacteria in water purification systems.

Novel Insights into Microbial Behavior Gleaned Using Microfluidics

“…In the areas of life sciences and biomedical engineering, the flexibility to design and control a local environment has been utilized to create increasingly sophisticated organ-on-a-chip devices (Bhatia and Ingber, 2014), which are enabling the study of organogenesis in controlled environments. A similar approach (Kunoh et al, 2020(Kunoh et al, , 2021 is being adopted in microbiology with soil-on-a-chip devices (Stanley et al, 2016) that attempt to mimic the environment or manage interkingdom interactions in a controlled manner, which will aid in the study of microbial ecology. We anticipate that the techniques and infrastructure needed to fabricate and operate microfluidic devices will become (Fukuda et al, 2021) more widely available, increasingly important, and make fundamental contributions to microbial ecology.…”

“…After clarifying single-filament dynamics, we investigated methods to control and modify the sheath, which may lead to practical methods for the management of bulking. We used microfluidics to exchange growth media quickly and easily in microfluidic devices, thereby permitting measurements of the effects of the abrupt limitation of different micronutrients on filament behavior (Kunoh et al, 2021). The findings obtained revealed that filament development under nutrient-deficient conditions may be classified into four general modes: (i) "normal", where the cell chain elongates without the appearance of intercellular gaps; (ii) "patchy", where large intercellular gaps appear within cell chains; (iii) "non-viable", where cell autolysis occurs before cell division may proceed; and (iv) "fragmented", where cell chains split into smaller chains (Fig.…”

Taxonomic characterization of Sphaerotilus microaerophilus sp. nov., a sheath-forming microaerophilic bacterium of activated sludge origin

“…Note that the subspecies affiliation of S. natans JCM 20382 is not determined (Gridneva et al 2011 ). Although these strains are not the type strains, they are commonly used as references to compare their phenotypic properties with those of related isolates (Sawayama et al 2011 ; Nott et al 2020 ; Kashiwabara et al 2021 ; Kunoh et al 2021 ). Growth under aerobic conditions was examined on GPPY agar (GPPY medium solidified with 1.5% agar) and APP medium at 25 °C for 10 days.…”

Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6

“…We considered the conjugation between WT* and S17-1 to be efficient when the cell surfaces of WT* cells were free from sheaths ( Figure 7 ). Since we previously reported that SP-6 cells become sheath-less when cultured in calcium-free conditions ( Kunoh et al, 2021 ), WT* cells cultured in routine MSVP were harvested and further cultured in MSVP lacking Ca (MSVP-Ca) for an additional 6 h (step M3) ( Figure 8A ). As expected, Figure 8B indicates that the conjugation efficiency with additional culturing in MSVP-Ca is approximately 2-fold higher than that without additional culturing (MSVP+Ca).…”

“…We previously reported that sheath structure plays a critical role in holding aligned cell chains of SP-6 ( Kunoh et al, 2020 ), and that calcium depletion frequently results in breakage of cell filaments of SP-6 due to failure in the production of sheath structures encasing the cell chains ( Kunoh et al, 2021 ). From these findings, we hypothesized that preculturing SP-6 cells in a calcium-lacking medium might enhance the accessibility of E. coli cells to SP-6 cells for conjugation, which results in efficient gene transfer and subsequent gene replacement of SP-6 cells.…”