Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

On the separation of 99mTcO4-, 99mTc-DTPA and 99mTc-citrate as marker species for the determination of Tc chemical forms in plant material using capillary zone electrophoresis

ABSTRACT

The present paper addresses the potential use of capillary zone electrophoresis (CZE) as an analytical tool in 99- technetium speciation studies. In order to optimise sampling, storage and analytical procedures, the three marker compounds ^99mTcO₄-, ^99mTc-DTPA and ^99mTc-citrate were synthesised and used in test-measurements with CZE. The results underline the superior separation power of the CZE technique, and indicate good CZE performance for the stable ^99mTcO₄- and ^99mTc-DTPA compounds. The data suggest that CZE may be used without problems for various Tc-compounds of intermediate mobilities. The specific data of ^99mTc-citrate suggest that with this marker compound a threshold lability is reached for the use of CZE in plant Tc-speciation studies. This result means that CZE cannot be used in analyses of Tc-compounds which are less stable than Tc-citrate. Future CZE work will comprise the synthesis and use of Tc-markers of intermediate mobilities and stabilities; furthermore, effects of marker matrices and the plant matrix on CZE performance will be investigated.     

Regulation of primary carbon metabolism in Kluyveromyces lactis

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called “Nonconventional Yeasts.” The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the “cell factory” concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.