Yoji Nakagawa

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.

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.

Identification and characterization of the pswP gene required for the parallel production of prodigiosin and serrawettin W1 in Serratia marcescens.

Serratia marcescens mutants defective in production of the red pigment prodigiosin and the biosurfactant serrawettin W1 in parallel were isolated by transposon mutagenesis of strain 274. Cloning of the DNA fragment required for production of these secondary metabolites with different chemical structures pointed out a novel open reading frame (ORF) named pswP. The putative product PswP (230 aa) has the distinct signature sequence consensus among members of phosphopantetheinyl transferase (PPTase) which phosphopantetheinylates peptidyl carrier protein (PCP) mostly integrated in the nonribosomal peptide synthetases (NRPSs) system. Since serrawettin W1 belongs to the cyclodepsipeptides, which are biosynthesized through the NRPSs system, and one pyrrole ring in prodigiosin has been reported as a derivative of L‐proline tethered to phosphopantetheinylated PCP, the mutation in the single gene pswP seems responsible for parallel failure in production of prodigiosin and serrawettin W1.

Bacterial wetting agents working in colonization of bacteria on surface environments

Abstract Bacteria are very small and highly susceptible to the effects of intermolecular forces. Especially, bacteria living on solid-air interfaces are under a strong influence from the surface tension of water. During studies of bacteria which prefer to live on surfaces, we noted that some species of bacteria (e.g. Serratia marcescens ) secrete large amounts of wetting agents (e.g. serrawettins). Therefore, we isolated mutants defective in the production of wetting agents and examined the physiological functions of these wetting agents by comparing the behavior of wild types and mutants on surface environments. In terms of accessibility to the water-repelling surfaces and spreading growth on solid media. mutants demonstrated inferior abilities in comparison with wild types. Thus, the ability of S. marcescens to form a giant fractal colony through nutrient-diffusion-limited growth processes was shown to be defective in the serrawettinless mutants. In the locomotion of flagellated bacteria on surfaces, the bacteria seem to overcome various restrictive intermolecular forces. In contrast to swimming in a liquid, a single bacterium alone was unable to translocate on a surface. By video-microscopic analyses, the cooperative multicellular behavior of bacteria was clearly demonstrated. The remarkable effects of wetting agents on such microbial swarming behavior on surfaces were also disclosed.

Surface-active exolipids : analysis of absolute chemical structures and biological functions

Abstract Absolute configuration of 3-hydroxy fatty acids in serrawettins, surface-active exolipids produced by Serratia marcescens , was elucidated by chiral column high-performance liquid chromatography. 3,5-Dinitroaniline or 3,5-dinitrophenyl urethane derivatives of hydroxy fatty acids were directly prepared from crude hydrolysates of the lipids and eluted separately through chiral columns (Enantio P2 or OA-3100) with specific eluents. In both serrawettin W1 and W2, d -3-hydroxydecanoic acid was determined as a major acyl group accompanied by a minor component d -3-hydroxy-dodecanoic acid. Many mutants defective in the production of serrawettins were isolated through screening by direct colony thin-layer chromatography and examined for altered behavioral phenotypes by various methods including digital analysis of colony patterns. Bacteria on surface environments seem to be living under strong surface tension of the surrounding water, and overcoming such circumstance by collaborative multicellular works and production of biosurfactants. Fractal colony growth of S. marcescens was shown as one such indication disclosing biological roles of the surface-active exolipid.

Resistance Mechanism of Chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1‐acetoxy, 3‐acetoxy and 1,3‐diacetoxy derivatives by thin‐layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE‐cellulose chromatography and gel filtration on Sephadex G‐150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000–80,000), optimum pH and heat stability.

Membrane filter (pore size, 0.22–0.45 μm; thickness, 150 μm) passing-through activity of Pseudomonas aeruginosa and other bacterial species with indigenous infiltration ability

Bacteria growing on MF-Millipore filters (thickness, 150 μm) passed through the underlying membrane by their infiltration activity. Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli passed through a 0.45-μm pore size filter within 48–96 h. Pseudomonas aeruginosa, Serratia marcescens, and Listeria monocytogenes passed through a 0.3-μm pore size filter. P. aeruginosa passed through a 0.22-μm pore size filter. The membranes which allowed passing-through of bacteria showed normal bubble point values in the integrity test. Studies with isogenic S. marcescens mutants indicated that flagellum-dependent motility or surface-active exolipid were important in the passing-through. P. aeruginosa PAO1 C strain defective in twitching motility was unable to pass through the 0.22-μm filter. Scanning electron microscopy showed bacteria passing-through the 0.22-μm filter. Millipore membrane filters having well-defined reticulate structures will be useful in the study of infiltration activity of microbes.

Serrawettins and Other Surfactants Produced by Serratia

Serrawettins are nonionic biosurfactants produced by Serratia marcescens. Three molecular species, serrawettin W1, cyclo(d-3-hydroxydecanoyl-l-seryl)2; W2, d-3-hydroxydecanoyl-d-leucyl-l-seryl-l-threonyl-d-phenylalanyl-l-isoleucyl lactone; and W3, cyclodepsipeptide composed of five amino acids and one dodecanoic acid, have been reported. Serratia rubidaea produces rubiwettin R1, linked d-3-hydroxy fatty acids and RG1, β-glucopyranosyl linked d-3-hydroxy fatty acids. These biosurfactants are produced mainly at 30°C, but not at 37°C, and secreted through extracellular vesicles on solid media. The contribution of the biosurfactants to spreading growth in surface environments has been determined, and it is prominent under nutrient-poor conditions. Analyses of S. marcescens mutants revealed the involvement of three novel genes for serrawettin W1 production. The gene pswP encodes a phosphopantetheinyl transferase group enzyme, swrW encodes a unimodular synthetase belonging to the nonribosomal peptide synthetase (NRPS) family, and hexS encodes a LysR-type transcriptional regulator working as a downregulator of Serratia exolipids and some exoenzymes. Autoinducer-dependent serrawettin W2 production has been elucidated by the finding of SwrI/SwrR (homolog of LuxI/LuxR) and N-acyl homoserine lactones in the study on quorum-sensing controlled-swarming of S. marcescens.

Rhamnolipid-dependent spreading growth of Pseudomonas aeruginosa on a high-agar medium : Marked enhancement under CO2-rich anaerobic conditions

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high‐agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O2‐depleted 8% CO2 condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid‐deficient rhlA mutant TR failed to exhibit spreading growth on a high‐agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard‐agar medium was unique to P. aeruginosa under CO2‐rich anaerobic conditions.