The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnal and gm€L homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnal and gm€LS after transfer from 30 "C to 43 "C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, off7 and g @ , resembled the pattern observed for the above genes, although maximum induction was observed earlier for off7 and grlpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for fcsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named odd, was shown not to be regulated by heat shock.

Background PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. Results In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. Conclusions In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA–amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORF b2 and ORF x, potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORF x and as a possibility for ORF b3 and ORF c2, which have ribosome-binding sites located more distant from their start codons. ORF b2 may be translationally fused with mhp at a low frequency. The mhp and mtp genes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

Mucor circinelloides (syn. racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes. We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M. circinelloides. In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor. We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M. circinelloides. In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium. However, during the dimorphic shift, i.e. during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold. These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions. Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching. Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.

Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu(-) phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.

The promoter of the Mucor circinelloides gpd1 gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd1P) was recently cloned and used for the production of recombinant proteins, such as the Aspergillus niger glucose oxidase 1 (GOX). This represents the first example of the application of a strong and regulated promoter from this fungus for recombinant protein production. The original 741-bp gpd1P promoter fragment conferred hexose-dependent expression of GOX in M. circinelloides. To understand the regulatory mechanisms involved in gpd1P-driven expression and to develop improved promoter fragments, deletion derivatives of gpd1P were constructed. These derivatives were fused to the A. niger gox1 gene and used to construct strains containing a single copy of the expression cassette. GOX activity was detected in strains containing the full-length gpd1P and also in strains containing a 713-bp or a 361-bp derivative. Expression levels for the 361-bp derivative were high and comparable, regardless of the carbon source used. This promoter represents a useful derivative for constitutive heterologous gene expression in M. circinelloides.