ABSTRACT
The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P(nisA) and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.
Subject(s)
Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases , Heat-Shock Proteins , Lactococcus lactis/metabolism , Micrococcal Nuclease , Periplasmic Proteins , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Brucella abortus/chemistry , Endonucleases/genetics , Endonucleases/metabolism , Lactococcus lactis/genetics , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomaviridae/genetics , Recombinant Proteins/metabolism , Rotavirus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
In bacterial communities one bacterium can influence the growth of other members of the population. These interactions may be based on nutritional factors or may occur via bacterial signaling molecules that are released in the medium. We present an example, showing that in addition to the above means of interactions, muramidases, enzymes that specifically cleave peptidoglycan chains, can also mediate interactions between bacteria. Using fluorescent in situ hybridization we demonstrate that Lactococcus lactis muramidase AcmA can hydrolyze the cell wall of Streptococcus thermophilus, without affecting viability. This intercellular activity of the lactococcal muramidase results in chain disruption of streptococci in vivo. Our data lead us to propose that chains can give growth advantages to streptococci in aerobic conditions.
Subject(s)
Bacterial Proteins/metabolism , Lactococcus lactis/enzymology , Muramidase/metabolism , Streptococcus/growth & development , Aerobiosis , Cell Wall/chemistry , Cell Wall/metabolism , Culture Media/chemistry , In Situ Hybridization, Fluorescence , Peptidoglycan/chemistry , Peptidoglycan/metabolism , Streptococcus/chemistryABSTRACT
Site-specific mutagenesis of the pentameric motif TGCAA within the regulatory region of the udp gene with coordinates -68 and -64 relative to the transcription initiation site was performed. Nine mutant promoters containing multiple nucleotide base-pair substitutions in this pentameric motif were isolated and characterized. One mutant contained a deletion of the C/G nucleotide pair in the -66 position. Isolated mutant promoters were cloned into a low-copy-number expression vector pJEL250 to determine the level of their expression, depending on the allelic state of cytR and cya genes. The level of CytR-dependent regulation of the udp gene and the ability to titrate the CytR repressor in vivo were shown to be drastically decreased in all mutant promoters isolated. On the basis of these results, it is concluded that the pentameric motif TGCAA plays a key role in binding the CytR repressor protein to the udp gene promoter.
Subject(s)
DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Repressor Proteins/genetics , Binding Sites/genetics , DNA Mutational Analysis , Escherichia coli Proteins , Promoter Regions, Genetic/genetics , Protein Binding , Transcriptional ActivationABSTRACT
The nucleotide sequence of a 1000-bp fragment of the Escherichia coli chromosome located between genes metE and udp and including the promoter region of the udp gene was determined. Multiple binding sites for the CytR and CRP proteins were identified outside the canonical udp gene promoter. A set of deletion variants with the truncated regulatory region of the udp gene was isolated based on plasmids pSKII and pJEL250. The level of CytR regulation of the udp gene was shown to depend on the size of the regulatory region relative to the transcription initiation site. On the basis of these data, it is concluded that additional binding sites for the CytR protein located in the regulatory region are functionally active in the regulation of udp gene expression. This conclusion has been confirmed by properties of the udp264 promoter mutant, which contains a deletion covering the main CytR binding site within the canonical promoter. Irrespective of the deletion, the expression of the udp gene in mutant udpP264 retains the dependence on the allelic state of the cytR gene. The CytR protein was shown to act as a transcription repressor or activator, depending on the configuration of the promoter and on the relative location and quantity of binding sites for CytR and CRP proteins.
