Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk.

In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow's milk (NWSc) for the production of caciocavallo and buffalo's milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (Lactococcus lactis subsp. lactis, Lactobacillus fermentum, Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) and five species in NWSc (Lc. lactis subsp. lactis, Enterococcus faecium, and S. thermophilus, Lb. helveticus, and Lb. delbrueckii), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on Lc. lactis subsp. lactis different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.

Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux.

Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both M. smegmatis and Mtb. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a MSMEG-3763 deletion mutant in M. smegmatis (∆MSMEG_3763). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.

Reliable identification of lactic acid bacteria by targeted and untargeted high-resolution tandem mass spectrometry.

Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity. In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/MS for LAB identification at genus, species and sub-species level by using the sequence regions 33-52 and 72-82 of the S16 ribosomal protein as proteotypic peptide markers. The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices.

Structural Characterization of the Lactobacillus Plantarum FlmC Protein Involved in Biofilm Formation.

Lactobacillus plantarum is one of the most predominant species in the human gut microbiota of healthy individuals. We have previously characterized some probiotic features of L. plantarum LM3, as the high resistance to different stress, the binding ability toward some extracellular matrix proteins and plasminogen and the immunomodulatory role of the surface expressed adhesin EnoA1. We have also identified the flmA, flmB and flmC genes, coding for putative proteins named FlmA, FlmB and FlmC, whose null mutations partially impaired biofilm development; the L. plantarum LM3⁻6 strain, carrying a deletion in flmC, showed a high rate of autolysis, supporting the hypothesis that FlmC might be involved in cell wall integrity. Here, we report the in-silico characterization of ΔTM-FlmC, a portion of the FlmC protein. The protein has been also expressed, purified and characterized by means of CD spectroscopy, ICP-mass and UHPLC-HRMS. The obtained experimental data validated the predicted model unveiling also the presence of a bound lipid molecule and of a Mg(II) ion. Overall, we provide strong evidences that ΔTM-FlmC belongs to the LytR-CpsA-Psr (LCP) family of domains and is involved in cell envelope biogenesis.

A Novel TetR-Like Transcriptional Regulator Is Induced in Acid-Nitrosative Stress and Controls Expression of an Efflux Pump in Mycobacteria.

Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its ortholog in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here, we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and used reporter assays to confirm that the MSMEG_3762/63/65 promoter was induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress.

Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

The Lactobacillus plantarum Eno A1 Enolase Is Involved in Immunostimulation of Caco-2 Cells and in Biofilm Development.

The role of probiotics in prevention and treatment of a variety of diseases is now well assessed. The presence of adhesive molecules on the cell surface of probiotics has been related to the ability to confer health benefit to the host. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is cell surface-expressed and is involved in binding with human fibronectin and plasminogen. By means of comparative analysis between L. plantarum LM3 (wild type) and its isogenic LM3-CC1 (ΔenoA1) mutant strain, here we show that EnoA1 affects the ability of this bacterium to modulate immune response as determined by analysis of expression of immune system molecules in Caco-2 cells. Indeed, we observed induction of TLR2 expression in cells exposed to L. plantarum LM3, while no induction was detectable in cells exposed to LM3-CC1. This difference was much less consistent when expression of TLR4 was determined in cells exposed to the two strains. Pro-inflammatory (IL-6) and anti-inflammatory cytokines (IL-10, TGF-ß), and the antimicrobial peptide HBD-2 were induced in Caco-2 cells exposed to L. plantarum LM3, while lower levels of induction were detected in cells exposed to LM3-CC1. We also analyzed the ability to develop biofilm of the two strains, and observed a decrease of about 65 % in the development of mature biofilm in LM3-CC1 compared to the wild type.

Identification and characterization of enolase as a collagen-binding protein in Lactobacillus plantarum.

Collagen is a target of pathogens for adhesion, colonization, and invasion of host tissue. Probiotic bacteria can mimic the same mechanism as used by the pathogens in the colonization process, expressing cell surface proteins that specifically interact with extracellular matrix component proteins. The capability to bind collagen is expressed by several Lactobacillus isolates, including some Lactobacillus plantarum strains. In this study we report the involvement of the L. plantarum EnoA1 alfa-enolase in type I collagen (CnI) binding. By adhesion assays, we show that the mutant strain LM3-CC1, carrying a null mutation in the enoA1 gene, binds to immobilized collagen less efficiently than wild type strain. CnI overlay assay and Elisa tests, performed on the purified EnoA1, show that this protein can bind collagen both under denaturing and native conditions. By using truncated recombinant enolase proteins, we also show that the region spanning from 73rd to the 140th amino acid residues is involved in CnI binding.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

Identification of binding sites of Lactobacillus plantarum enolase involved in the interaction with human plasminogen.

The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface.

CcpA and three newly identified proteins are involved in biofilm development in Lactobacillus plantarum.

The aim of this study was to identify genes involved in biofilm development in the probiotic lactic acid bacterium Lactobacillus plantarum. The ability of L. plantarum LM3 and of some derivative mutant strains to form biofilm has been investigated. Biofilm microtitre plate assays showed that L. plantarum LM3-2, carrying a null mutation in the ccpA gene, coding the CcpA master regulator, was partially impaired in biofilm production compared to wild type (LM3). Moreover, we found three genes in the L. plantarum genome, hereby named flmA, flmB, and flmC, whose deduced amino acid sequences show significant identity with the Streptococcus mutans BrpA (biofilm regulatory protein A). We investigated the role of FlmA, FlmB, and FlmC in biofilm formation by isolating strains carrying null mutations in the corresponding genes. Our results suggest involvement of the Flm proteins in biofilm development. Moreover, transcriptional studies show that expression of flmA, flmB, and flmC is under the control of CcpA. These results, together with the reduced ability of LM3-2 (ccpA1) to form biofilm, strongly suggest a positive role of the master regulator CcpA in biofilm development.

Inactivation of ccpA and aeration affect growth, metabolite production and stress tolerance in Lactobacillus plantarum WCFS1.

The growth of Lactobacillus plantarum WCFS1 and of its ΔccpA ery mutant, WCFS1-2, was compared in batch fermentations in a complex medium at controlled pH (6.5) and temperature (30°C) with or without aeration, in order to evaluate the effect of ccpA inactivation and aeration on growth, metabolism and stress resistance. Inactivation of ccpA and, to a lesser extent, aeration, significantly affected growth, expression of proteins related to pyruvate metabolism and stress, and tolerance to heat, oxidative and cold/starvation stresses. The specific growth rate of the mutant was ca. 60% of that of the wild type strain. Inactivation of ccpA and aerobic growth significantly affected yield and production of lactic and acetic acid. Stationary phase cells were more stress tolerant than exponential phase cells with little or no effect of inactivation of ccpA or aeration. On the other hand, for exponential phase cells inactivation of ccpA impaired both heat stress and cold/starvation stress, but increased oxidative stress tolerance. For both strains, aerobically grown cells were more tolerant of stresses. Evidence for entry in a viable but non-culturable status upon prolonged exposure to cold and starvation was found. Preliminary results of a differential proteomic study further confirmed the role of ccpA in the regulation of carbohydrate catabolism and class I stress response genes and allow to gain further insight on the role of this pleiotropic regulator in metabolism and stress. This is the first study in which the impact of aerobic growth on stress tolerance of L. plantarum is evaluated. Although aerobic cultivation in batch fermentations does not improve growth it does improve stress tolerance, and may have significant technological relevance for the preservation of starter and probiotic cultures.

Expression of the Lactobacillus plantarum malE gene is regulated by CcpA and a MalR-like protein.

Lactobacillus plantarum is commonly used in the food industry as a starter in various fermentations, especially in vegetable fermentations, in which starch is a common substrate. This polysaccharide, which is obtained from potatoes or corn and is hydrolysed mainly to maltose and glucose by acids or enzymes, is commercially used for the production of lactate by lactic acid fermentation. In this study, we describe the regulation of malE gene expression in L. plantarum. This gene, located in a 7-gene cluster, probably organized in an operon, encodes a putative maltose/maltodextrin-binding protein. We studied the expression of malE in L. plantarum LM3 (wild type) and in LM3-2 (ccpA1), which carries a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA). In the presence of glucose, expression of the MalE protein was higher in the mutant strain as compared to that in the wild-type strain. Transcription of the malE gene was induced by maltose and regulated by a CcpA-mediated carbon catabolite repression. Further, we isolated strains carrying mutations in 2 genes, lp_0172 and lp_0173, whose deduced amino acid sequences share significant identity with MalR, a regulator of the maltose operon in several gram-positive bacteria. A double mutant exhibited glucose-insensitive malE transcription, while absence of the functional Lp_0172 open reading frame had no effect on malE expression.

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein.

BACKGROUND: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. RESULTS: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. CONCLUSION: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein.

CcpA affects expression of the groESL and dnaK operons in Lactobacillus plantarum.

BACKGROUND: Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. During industrial processes changes in temperature may represent an environmental stress to be overcome by starters and non-starters LAB. Studies on adaptation to heat shock have shown the involvement of the chaperon system-proteins in various gram-positive bacteria. The corresponding operons, namely the dnaK and groESL operons, are controlled by a negative mechanism involving the HrcA repressor protein binding to the cis acting element CIRCE. RESULTS: We studied adaptation to heat shock in the lactic acid bacterium Lactobacillus plantarum. The LM3-2 strain, carrying a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA), showed a lower percent of survival to high temperature with respect to the LM3 wild type strain. Among proteins differentially expressed in the two strains, the GroES chaperon was more abundant in the wild type strain compared to the mutant strain under standard growth conditions. Transcriptional studies showed that class I heat shock operons were differentially expressed upon heat shock in both strains. Indeed, the dnaK and groESL operons were induced about two times more in the LM3 strain compared to the LM3-2 strain. Analysis of the regulatory region of the two operons showed the presence of cre sequences, putative binding sites for the CcpA protein. CONCLUSION: The L. plantarum dnaK and groESL operons are characterized by the presence of the cis acting sequence CIRCE in the promoter region, suggesting a negative regulation by the HrcA/CIRCE system, which is a common type of control among the class I heat shock operons of gram-positive bacteria. We found an additional system of regulation, based on a positive control exerted by the CcpA protein, which would interact with cre sequences present in the regulatory region of the dnaK and groESL operons. The absence of the CcpA protein results in a lower induction of the chaperon coding operons, with a consequent lower percent of survival of the LM3-2 mutant strain population with respect to the wild type when challenged with a heat insult.

Involvement of pyruvate oxidase activity and acetate production in the survival of Lactobacillus plantarum during the stationary phase of aerobic growth.

In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.

Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum.

The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized. Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter. poxB mRNA expression was strongly induced by aeration and was repressed by glucose. Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted. Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region. The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production. Constitutive overproduction of PoxB in L. plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions. The DeltapoxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose). In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production. In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.

Mutational analysis of the bglH catabolite-responsive element (cre) in Lactobacillus plantarum.

Catabolite repression of the Lactobacillus plantarum bglH gene is mediated by the catabolite control protein A (CcpA). Here we show that the binding site for the protein is a catabolite-responsive element (cre) sequence overlapping the start site of transcription. Two single and one double base substitutions in the cre sequence caused derepression of a plasmid-borne bglH-cat transcriptional fusion in L. plantarum cells grown on glucose. Analysis of the single mutations indicates that the G and C nucleotide residues in positions 2 and 13, respectively, of the 14-bp cre sequence are required for catabolite repression.

Membrane topology analysis of the Bacillus subtilis BofA protein involved in pro-sigma K processing.

The Bacillus subtilis BofA protein is involved in regulation of pro-sigma K processing in the mother cell during the late stages of sporulation. A computer analysis of the BofA amino acid sequence indicates that it is an integral membrane protein. To determine the membrane topology of the protein, a series of gene fusions of bofA with lacZ or phoA reporter genes in Escherichia coli were analysed. A BofA topological model with two membrane-spanning segments, and with the N- and the C-terminal domains located in the region between the inner and outer membranes surrounding the forespore is presented. The analysis of different modifications of the last five amino acid residues of the BofA protein, obtained by PCR site-directed mutagenesis, suggests a possible role of the C-terminal domain in the regulation of pro-sigma K processing.