Tohey Matsuyama

Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans

The nematode Caenorhabditis elegans is present in soils and composts, where it can encounter a variety of microorganisms. Some bacteria in these rich environments are innocuous food sources for C. elegans, whereas others are pathogens. Under laboratory conditions, C. elegans will avoid certain pathogens, such as Serratia marcescens, by exiting a bacterial lawn a few hours after entering it. By combining bacterial genetics and nematode genetics, we show that C. elegans specifically avoids certain strains of Serratia based on their production of the cyclic lipodepsipentapeptide serrawettin W2. Lawn-avoidance behavior is chiefly mediated by the two AWB chemosensory neurons, probably through G protein-coupled chemoreceptors, and also involves the nematode Toll-like receptor gene tol-1. Purified serrawettin W2, added to an Escherichia coli lawn, can directly elicit lawn avoidance in an AWB-dependent fashion, as can another chemical detected by AWB. These findings represent an insight into chemical recognition between these two soil organisms and reveal sensory mechanisms for pathogen recognition in C. elegans.

Interface growth and pattern formation in bacterial colonies

Bacterial species Bacillus subtilis is known to exhibit various colony patterns, depending on the substrate softness and nutrient concentration. We have examined the self-affinity and roughness exponent α for growing interfaces of colonies which belong to regions B (Eden-like) and D (disk-like) in the morphological phase diagram of this species. We have obtained α≃0.78 and 0.50 in the regions B and D, respectively, and found that the difference of α values can be attributed to that of microscopic growth mechanisms of colony interfaces. We have also proposed a unified model which produces characteristic colony patterns observed in our experiments. It is a diffusion–reaction-type model for the population density of bacterial cells and the concentration of nutrient. The essential assumption is that there exist two types of bacterial cells; active and inactive. Our model is found to be able to globally reproduce all the colony patterns in the phase diagram.

Extracellular Vesicle Formation and Biosurfactant Production by Serratia marcescens

Summary: Pigmented and non-pigmented strains of Serratia marcescens produced extracellular vesicles and had wetting activity when grown at 30°C but not at 37°C. Light microscopy showed that the red pigment was present in vesicles and intracellular granules. Electron microscopy revealed the presence of vesicles surrounded by the bacterial membrane. Three lipids having the wetting activity, W1, W2 and W3, were isolated by thin-layer chromatography of lipids from different strains of S. marcescens. Dispersions of the isolated wetting agents had small contact angles on a polystyrene surface and the ability to lower surface tension. Wetting agent W1 was the aminolipid serratamolide. Wetting agents W2 and W3 were also aminolipids but were shown to be different from serratamolide by chemical analyses. Wetting agent and prodigiosin (in a pigmented strain) were the main lipids of isolated vesicles.

Formation of colony patterns by a bacterial cell population

Bacterial species Bacillus subtilis is known to exhibit various colony patterns, such as diffusion-limited aggregation (DLA)-like, compact Eden-like, dense branching morphology (DBM)-like, concentric ring-like and disk-like, depending on the substrate softness and nutrient concentration. We have established the morphological diagram of colony patterns, and examined and characterized both macroscopically and microscopically how they grow. For instance, we have found that there seem to be two kinds of bacterial cells; active and inactive cells, the former of which drive colony interfaces outward. The active cells are particularly distinguished from the inactive ones at the tips of growing branches of a DBM-like colony as the characteristic fingernail structure. We have also found that the concentric ring-like colony is formed as a consequence of alternate repetition of advancing and resting of the growing interface which consists of active cells. Based on our observations, we have constructed a phenomenological but unified model which produces characteristic colony patterns. It is a reaction–diffusion type model for the population density of bacterial cells and the concentration of nutrient. The essential assumption is that there exist two types of bacterial cells; active cells that move actively, grow and perform cell division, and inactive ones that do nothing at all. Our model is found to be able to reproduce globally all the colony patterns seen in the experimentally obtained morphological diagram, and is phenomenologically quite satisfactory.

Dynamic Aspects of the Structured Cell Population in a Swarming Colony of Proteus mirabilis

Proteus mirabilis forms a concentric-ring colony by undergoing periodic swarming. A colony in the process of such synchronized expansion was examined for its internal population structure. In alternating phases, i.e., swarming (active migration) and consolidation (growth without colony perimeter expansion), phase-specific distribution of cells differing in length, in situ mobility, and migration ability on an agar medium were recognized. In the consolidation phase, the distribution of mobile cells was restricted to the inner part of a new ring and a previous terrace. Cells composing the outer part of the ring were immobile in spite of their ordinary swimming ability in a viscous solution. A sectorial cell population having such an internal structure was replica printed on fresh agar medium. After printing, a transplant which was in the swarming phase continued its ongoing swarming while a transplanted consolidation front continued its scheduled consolidation. This shows that cessation of migration during the consolidation phase was not due to substances present in the underlying agar medium. The ongoing swarming schedule was modifiable by separative cutting of the swarming front or disruption of the ring pattern by random mixing of the pattern-forming cell population. The structured cell population seemed to play a role in characteristic colony growth. However, separation of a narrow consolidation front from a backward area did not induce disturbance in the ongoing swarming schedule. Thus, cells at the frontal part of consolidation area were independent of the internal cell population and destined to exert consolidation and swarming with the ongoing ordinary schedule.

Serratia marcescens Gene Required for Surfactant Serrawettin W1 Production Encodes Putative Aminolipid Synthetase Belonging to Nonribosomal Peptide Synthetase Family

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin‐less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi‐modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni‐modular NRPS specifying only L‐serine. Possible steps in this simple uni‐modular NRPS for biosynthesis of serrawettin W1 [cyclo‐(D‐3‐hydroxydecanoyl‐L‐seryl)2] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi‐modular NRPS) of Brevibacillus brevis.

Experimental tinea pedis induced by non-abrasive inoculation of Trichophyton mentagrophytes arthrospores on the plantar part of a guinea pig foot

Arthrospores of Trichophyton mentagrophytes were inoculated on to the plantar part of a guinea pig foot by a newly devised non-abrasive method. Anthropophilic and zoophilic isolates required inocula of 280 and 80 arthrospores to infect 50% of inoculated feet, but much larger inocula (5 X 10(4)) were used to establish infection consistently in all feet. Anthropophilic isolate NTM-105 invaded only the upper two-thirds of the horny layer and induced no inflammatory responses. On the other hand, zoophilic isolate SM-110 invaded the whole horny layer and provoked strong inflammatory responses and clinical manifestations. Although the histological features and modes of fungal spreading in the guinea pig skin were quite different between anthropophilic and zoophilic isolate infections, infecting fungi were always recognized in the stratum corneum of all inoculated feet throughout the observation period longer than 6 months. Thus, two types of persistent infections with T. mentagrophytes were established as a guinea pig model of tinea pedis.

Transcriptional Downregulator HexS Controlling Prodigiosin and Serrawettin W1 Biosynthesis in Serratia marcescens

Serratia marcescens has been known as a temperature‐dependent producer of two chemically different exolipids (red pigment prodigiosin and biosurfactant serrawettin W1) in parallel. During genetic investigation of such control mechanisms, mini‐Tn5 insertional mutant Tan1 overproducing these exolipids was isolated. The gene concerning such disregulation was identified as hexS by DNA cloning followed by sequencing and homology analysis of the presumed product with 314 amino‐acids. The product HexS was the homologue of HexA of Erwinia carotovora ssp. carotovora and classified as a transcriptional regulator belonging to LysR family. By RT‐PCR analysis, the hexS mutant was shown to over‐transcribe the pigA gene (the first gene of the pig cluster involved in prodigiosin synthesis) and the swrW gene encoding serrawettin W1 synthetase belonging to the nonribosomal peptide synthetase family. In contrast, transcription of the pswP gene encoding phosphopantetheinyl transferase in Tan1 was in the level of parent strain 274. Purified protein encoded in his6‐hexS demonstrated binding activity to DNA fragments of the upstream region of pigA and swrW genes and not to that of the pswP gene. S. marcescens strain 274 transformed with a low‐copy plasmid carrying hexS demonstrated reduced production of prodigiosin and serrawettin W1, and reduced activity of exoenzymes (protease, chitinase, and DNase) except phospholipase C. Possible generation of virulent S. marcescens by derepression or mutation of the hexS gene in infected tissues or ex vivo environments was suggested.

Characteristic infectivity of Sporothrix schenckii to mice depending on routes of infection and inherent fungal pathogenicity

Isolates of Sporothrix schenckii were examined for their infectivity in BALB/c mice. The mice were injected with yeast forms of S. schenckii isolates differing in clinical source (human cutaneous lesions and pulmonary lesions), and fungal growth was determined at intervals in the footpad and visceral organs. After subcutaneous injection of approximately 10 colony forming units (cfu) of S. schenckii into the footpad, locally restricted fungal infection developed gradually. At the peak of the infection (3-4 weeks post-inoculation), viable fungal counts reached 102-106 cfu/footpad. Dissemination to other tissues and visceral organs was not observed. After intravenous or intraperitoneal injection of 106 cfu of yeast forms, three of four isolates from cutaneous sporotrichosis were unable to establish infection and were eliminated from blood and visceral organs. The development of systemic infection was observed only with S. schenckii isolates obtained from the human lung lesion. Thus, inherent properties of each clinical isolate and routes of infection were shown to be critical for the establishment of systemic infection in spite of the remarkably strong infectivity of S. schenckii to the cutaneous tissue.

Experimental Investigation on the Formation of Dense-Branching-Morphology-Like Colonies in Bacteria

We have investigated experimentally the pattern formation of bacterial colonies. We have especially examined DBM (dense-branching-morphology)-like colonies of bacterial species Bacillus subtilis . It was found from microscopic observations that many active bacterial cells collect and make a group on the tip of each growing branch. Branches repel each other and split sometimes as they grow outward. It was confirmed macroscopically that both averaged branch width and averaged branch gap decrease systematically when increasing the nutrient concentration C n , while their ratio remains unchanged with the approximate value of one. It was also found that the distribution of branch lengths is very close to exponential, suggesting that the tip-splitting of branches takes place at random.