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Lactobacillus casei transports glucose preferentially by a mannose-class phosphoenolpyruvate sugar phosphotransferase system (PTS). The genomic analysis of L. casei allowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded by manO), homologues of which are also present in man clusters that encode glucose/mannose transporters in other Gram-positive bacteria. The L. casei man operon is constitutively expressed into a manLMNQ messenger, but an additional manQ transcript was also detected. Upstream of the man operon, two genes (upsR and upsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of either upsR or upsA did not affect manLMNO transcription, and had no effect on glucose uptake. Cells carrying a manO deletion transported glucose at a rate similar to that of the wild-type strain. By contrast, a manM disruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of the man genes. In addition, the manM mutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes in L. casei.

A Lactobacillus casei strains expressing extracellular or cell-wall-attached anti-VP8 single-chain antibodies (scFv) that may be useful to deliver passive immunity against rotavirus was discussed. Results showed that the secreted scFv had a correct folding that would retain its biological activity and demonstrated the potential of L. casei to be engineered to express such antibodies.

In low-G + C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactebacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T3071, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.

Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and El proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and El proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr ([His.sub.6]) was purified from wild-type and a Ser46/GIn mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orr2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an [Ell.sup.Nag] element of the PTS, as yet uncharacterized. In addition, GlcNAc-6phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.

Research has been conducted on the expression regulation of the lactone operon in Lactobacillus casei by the antiterminator LacT. Results indicate that phosphate group transfer from the PTS elements could have different affinity for the conserved domains of LacT.

The lactose operon of Lactobacillus casei could be exploited to genetically engineer the bacterium to express genes for industrial, clinical, and veterinary uses. Researchers have used the operon to express the E. coli gusA gene and the L. lactis ilvBN gene in L. casei.

Results reveal that HPr kinase/P-Ser-HPr phosphatase of gram-positive bacteria is a regulator protein and controls catabolic expression. Data also point out that the bifunctional enzyme may not be involved in the inducer expulsion regulatory process.

Research has been conducted on the genes that encode L-sorbose metabolism of Lactobacillus casei. The sequence analysis of this gene has been carried out.

A DNA fragment from Lactobacillus casei that restores growth to Escherichia coli and Salmonella typhimurium ptsH mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system was isolated. The mutants do not have the HPr protein. Sequencing of the cloned fragment indicated the absence of ptsH homologues. Instead, the complementation ability was found in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria.

The genes involved in the arginine deiminase (ADI) pathway of Lactobacillus sake were cloned and characterized to examine the regulation of arginine catabolism. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI, carbamate kinase, ornithine transcarbamoylase and a putative carrier. A putative transaminase-encoding gene was also located in this region. Construction of mutants through genetic disruption in all of the encoded enzymes showed that the enzymes are necessary for correct functioning of the pathway.

Cloning and sequencing of the chromosomal ccpA gene from Lactobacillus casei ATCC 393 were conducted. The findings indicate that deletion of the cre site or the presence of the ccpA mutation ended the catabolite repression of a lacp::gusA fusion. This result underscores the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.

A phosphoenolpyruvate:mannose phosphotransferase system (mannose-PTS) was found to be involved in glucose and mannose transport in Lactobacillus curvatus. However, no glucose-specific PTS activity could be detected. Sequence analysis of a 2*1 kb DNA fragment amplified by PCR from the L. curvatus genome revealed four open reading frames which could encode proteins similar to PTS transporters EIIA, EIIB, EIIC and EIID of the mannose class. It was also found that the sugar specificity pattern corresponds to an EII(super Man)-type of transporter and that fructose is not transported by the system.

Glucose transport in Lactobacillus casei ATCC 393 strain is phosphoenolpyruvate (PEP) mannose-dependent. A proton-motive-force-dependent glucose permease is present in the mutant that is resistant to glucose. The complex IIABC(man) catalyzes repression of lactose and ribose assimilation genes by glucose and mannose.

Functional and regulatory molecular and genetic analyses are used for studying the sorbitol operon from Lactobacillus casei. The gutM mutant has shown a reduced gut operon expression and sorbitol uptake, thus indicating a regulatory role for the gene encoding regulatory protein GutM.

A study was conducted on the lactose-inducible system using Lactobacillus casei lactose operon promoter. It was observed that the recombination of Lactobacillus casei cells played a very important role in the production of human papillomavirus type 16LI virus-like particles, and possibility for development of new live mucosal prophylactic vaccines.

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar. doi: 10.1128/JB.00076-10

Studies the existence of a true phosphofructokinase (PFK) in Bacillus sphaericus. Detection of PFK activity in Bacillus sphaericus; Amplification and sequencing of the pfk gene of Bacillus sphaericus 2362.

Shows that the sorbitol-6-phosphate dehydrogenase genes are required for Lactobacillus casei growth on L-sorbose and D-sorbitol. Biochemical and transcriptional analyses of wild-type and mutant strains; DNA manipulation and sequencing; Chromosomal inactivation of genes.

Describes the sixth gene in the arginine deiminase gene cluster of the Lactobacillus sakei. Sequence analysis of the arcR gene; Tendency for the arcR gene to be coordinately transcribed with the other arc genes.

Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IoIR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23. [ABSTRACT FROM AUTHOR]