Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis.

The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.

Inducible gene expression systems in Lactococcus lactis.

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described in L. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems in Lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.

Bacteriophage-triggered defense systems: phage adaptation and design improvements.

A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene. Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay.

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.