Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.     

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.     

Evidence for geographic isolation and signs of endemism within a protistan morphospecies

The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of "Spumella-like" flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.     

Population dynamics within a microbial consortium during growth on diesel fuel in saline environments

The diversity and dynamics of a bacterial community extracted from an exploited oil field with high natural soil salinity near Comodoro Rivadavia in Patagonia (Argentina) were investigated. Community shifts during long-term incubation with diesel fuel at four salinities between 0 and 20% NaCl were monitored by single-strand conformation polymorphism community fingerprinting of the PCR-amplified V4-V5 region of the 16S rRNA genes. Information obtained by this qualitative approach was extended by flow cytometric analysis to follow quantitatively the dynamics of community structures at different salinities. Dominant and newly developing clusters of individuals visualized via their DNA patterns versus cell sizes were used to identify the subcommunities primarily involved in the degradation process. To determine the most active species, subcommunities were separated physically by high-resolution cell sorting and subsequent phylogenetic identification by 16S rRNA gene sequencing. Reduced salinity favored the dominance of Sphingomonas spp., whereas at elevated salinities, Ralstonia spp. and a number of halophilic genera, including Halomonas, Dietzia, and Alcanivorax, were identified. The combination of cytometric sorting with molecular characterization allowed us to monitor community adaptation and to identify active and proliferating subcommunities.     

Über ein inkretorisches Cerebralorgan bei Lumbriciden,sowie Beschreibung eines verwandten Organs bei drei neuen Lycastis-Arten

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