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Lactococcus lactis is a Gram-positive bacterium that is widely used as a cell factory for the expression of heterologous proteins that are relevant in the pharmaceutical and nutraceutical fields. The signal peptide of the major secreted protein of L. lactis, Usp45, has been employed extensively in engineering strategies to secrete proteins of interest. However, the biological function of Usp45 has remained obscure despite more than 25 years of research. Studies on Usp45 homologs in other Gram-positive bacteria suggest that Usp45 may play a role in cell wall turnover processes. Here, we show the effect of inactivation and overexpression of the usp45 gene on L. lactis growth, phenotype, and cell division. Our results are in agreement with those obtained in streptococci and demonstrate that the L. lactis Usp45 protein is essential for proper cell division. We also show that the usp45 promoter is highly activated by galactose. Overall, our results indicate that Usp45 mediates cell separation, probably by acting as a peptidoglycan hydrolase. IMPORTANCE The cell wall, composed mainly of peptidoglycan, is key to maintaining the cell shape and protecting the cell from bursting. Peptidoglycan degradation by peptidoglycan hydrolysis and autolysins occurs during growth and cell division. Since peptidoglycan hydrolases are important for virulence, envelope integrity, and regulation of cell division, it is valuable to investigate their function and regulation. Notably, PcsB-like proteins such as Usp45 have been proposed as new targets for antimicrobial drugs and could also be target for the development of food-grade suicide systems. In addition, although various other expression and secretion systems have been developed for use in Lactococcus lactis, the most-used signal peptide for protein secretion in this bacterium is that of the Usp45 protein. Thus, elucidating the biological function of Usp45 and determining the factors affecting its expression would contribute to optimize several applications.

Lactococcus lactis is a Gram-positive bacterium widely used as starter culture for the production of different dairy products, especially a large variety of cheeses. Infection of lactococcal starter cultures by bacteriophages is one of the major causes of fermentation failure and often leads to production halt. Lactococcal bacteriophages belonging to the c2, 936 and P335 species are the most commonly isolated in dairy plants and have been extensively investigated in the past three decades. Information regarding bacteriophages belonging to less commonly isolated species is, on the other hand, less extensive, although these phages can also contribute to starter culture infection. Here we report the nucleotide sequence of the newly isolated L. lactis phage CHPC971, belonging to the rare 1706-species of lactococcal phages. We investigated the nature of the host receptor recognized by the phage and collected evidence strongly suggesting that it binds to a specific sugar moiety in the cell wall pellicle of its host. An in silico analysis of the genome of phage CHPC971 identified the hypothetical genes involved in receptor binding.Importance: Gathering information on how lactococcal bacteriophages recognize their host and proliferate in the dairy environment is of vital importance for the establishment of proper starter culture rotation plans, and to avoid fermentation failure and consequent great economic losses for dairy industries. We provide strong evidence on the type of receptor recognized by a newly isolated 1706-type lactococcal bacteriophage, increasing the knowledge on phage-host interactions relevant to dairying. This information can help to prevent phage infection events that, so far, are hard to predict and avoid.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e., Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter aerogenes ) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens. IMPORTANCE Lantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

Lactococcus lactis subsp. cremoris strains typically carry many dairy niche-specific adaptations. During adaptation to the milk environment these former plant strains have acquired various pseudogenes and insertion sequence elements indicative of ongoing genome decay and frequent transposition events in their genomes. Here we describe the reactivation of a silenced plant sugar utilization cluster in an L. lactis MG1363 derivative lacking the two main cellobiose transporters, PtcBA-CelB and PtcBAC, upon applying selection pressure to utilize cellobiose. A disruption of the transcriptional repressor gene llmg_1239 by an insertion sequence (IS) element allows expression of the otherwise silent novel cellobiose transporter Llmg_1244 and leads to growth of mutant strains on cellobiose. Llmg_1239 was labeled CclR, for c ellobiose cl uster r epressor. IMPORTANCE Insertion sequences (ISs) play an important role in the evolution of lactococci and other bacteria. They facilitate DNA rearrangements and are responsible for creation of new genetic variants with selective advantages under certain environmental conditions. L. lactis MG1363 possesses 71 copies in a total of 11 different types of IS elements. This study describes yet another example of an IS-mediated adaptive evolution. An integration of IS 981 or IS 905 into a gene coding for a transcriptional repressor led to activation of the repressed gene cluster coding for a plant sugar utilization pathway. The expression of the gene cluster allowed assembly of a novel cellobiose-specific transporter and led to cell growth on cellobiose.

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins., This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and the Plan for Science, Technology and Innovation 2013–2017, funded by the European Regional Development Fund and the Principality of Asturias (GRUPIN14-137). D.M.L. and B.D.R. were the beneficiaries of JAE DOC contracts (CSIC).

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d -alanine in the lantibiotic lacticin 3147. The conversion of l -serine into d -alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis . We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.

In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis , natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis , natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX -driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK -harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo , and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis , S. pneumoniae , and Lactococcus lactis , using promoter- gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa . The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA2mob required higher HA temperatures for efficient germination than spores lacking spoVA2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers.

The Lactococcus lactis laboratory strain MG1363 has been described to be unable to utilize lactose. However, in a rich medium supplemented with lactose as the sole carbon source, it starts to grow after prolonged incubation periods. Transcriptome analyses showed that L. lactis MG1363 Lac + cells expressed celB , encoding a putative cellobiose-specific phosphotransferase system (PTS) IIC component, which is normally silent in MG1363 Lac − cells. Nucleotide sequence analysis of the cel cluster of a Lac + isolate revealed a change from one of the guanines to adenine in the promoter region. We showed here that one particular mutation, taking place at increased frequency, accounts for the lactose-utilizing phenotype occurring in MG1363 cultures. The G-to-A transition creates a −10 element at an optimal distance from the −35 element. Thus, a fully active promoter is created, allowing transcription of the otherwise cryptic cluster. Nuclear magnetic resonance (NMR) spectroscopy results show that MG1363 Lac + uses a novel pathway of lactose utilization.

pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis strains with and without pBL1 were compared. A discrete response was observed, with a total of 10 genes showing significantly changed expression. Upregulation of the lactococcal oligopeptide uptake (opp) system was observed, which was likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Strikingly, celB, coding for the membrane porter IIC of the cellobiose phosphoenolpyruvate-dependent phosphotransferase system (PTS), and the upstream gene llmg0186 were downregulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1, and MG1363 ΔcelB grown in chemically defined medium (CDM) containing cellobiose confirmed slower growth of MG1363/pBL1 and MG1363 ΔcelB, while no differences were observed with growth on glucose. The presence of pBL1 shifted the fermentation products toward a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cells growing on cellobiose as determined by highpressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugar pools were lower, which could reflect rerouting of precursors toward the production of structural or storage polysaccharides. Moreover, cells growing slowly on cellobiose and those lacking celB were more tolerant to Lcn972 than cellobiose-adapted cells. Thus, downregulation of celB could help to build up a response against the antimicrobial activity of Lcn972, enhancing self-immunity of the producer cells. © 2011, American Society for Microbiology., P. Gaspar was supported by a Fundação para a Ciência e a Tecnologia (FCT) postdoctoral grant (SFRH/BPD/31251/2006), and C. Roces holds a JAEPre-CSIC (Spain) predoctoral fellowship. B. Martínez and A. R. Neves gratefully acknowledge the Ministerio de Ciencia e Innovación (MCINN) and Conselho de Reitores das Universidades Portuguesas (CRUP) for the Luso-Spanish Integrated Action (HP2008-0042; Pt, E-67/09). This work was supported by grants BIO2007-65061 and BIO2010-17414 (MCINN-Spain) and in part by FCT grants PTDC/SAU-MII/100964/2008 and PTDC/BIA-MIC/099963/2008. The NMR spectrometers are part of the National NMR Network (REDE/1517/RMN/2005), supported by “Programa Operacional Ciência e Inovação (POCTI) 2010” and FCT.

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis , with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway ( gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway ( lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE , encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT , but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

In previous studies, we have shown that direct protein-protein interaction between the two regulators ArgR and AhrC in Lactococcus lactis is required for arginine-dependent repression of the biosynthetic argC promoter and the activation of the catabolic arcA promoter. Here, we establish the global ArgR and AhrC regulons by transcriptome analyses and show that both regulators are dedicated to the control of arginine metabolism in L. lactis .

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the “reading frame” of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the “phasing” of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin ( myc ) and surfactin ( srfA ) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated.

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.

Achieving efficient heterologous protein production and secretion by Bacillus subtilis is an attractive prospect, although often disappointingly low yields are reached. The expression of detoxified Clostridium perfringens β-toxin (β-toxoid) is exemplary for this. Although β-toxin can be efficiently expressed and secreted by Bacillus subtilis , the genetically detoxified, and industrially interesting, β-toxoid variant is difficult to obtain in high amounts. To optimize the expression of this putative vaccine component, we studied the differences in the global gene regulation responses of B. subtilis to overproduction of either β-toxin or β-toxoid by transcriptomics. A clear difference was the upregulation of the CssRS regulon, known to be induced upon secretion stress, when β-toxoid is produced. YkoJ, a protein of unknown function, was also upregulated, and we show that its expression is dependent on cssS . We then focused on the heterologous protein itself and found that the major secretion bottleneck can be traced back to a single amino acid substitution between the β-toxin and the β-toxoid, which results in the rapid degradation of β-toxoid following secretion across the cytoplasmic membrane. In contrast to β-toxin, β-toxoid protein is more prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with point mutations. Our results show that although the host can be adapted in many ways, the intrinsic properties of a heterologous protein can play a decisive role when optimizing heterologous protein production.

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis , this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli , induces secretion stress in B. subtilis . Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis . The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis . Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30°C increased approximately 2.5-fold following a shift from 30 to 10°C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via α,γ elimination to form volatile aroma compounds.

Low-temperature adaptation and cryoprotection were studied in the thermophilic lactic acid bacterium Streptococcus thermophilus CNRZ302. S. thermophilus actively adapts to freezing during a pretreatment at 20°C, resulting in an approximately 1,000-fold increased survival after four freeze-thaw cycles compared to mid-exponential-phase cells grown at an optimal temperature of 42°C. No adaptation is observed when cells are exposed to a temperature (10°C) below the minimal growth temperature of the strain (just below 15°C). By two-dimensional gel electrophoresis several 7-kDa cold-induced proteins were identified, which are the major induced proteins after a shift to 20°C. These cold shock proteins were maximally expressed at 20°C, while the induction level was low after cold shock to 10°C. To confirm the presence of csp genes in S. thermophilus , a PCR strategy was used which yielded products of different sizes. Sequence analysis revealed csp -like sequences that were up to 95% identical to those of csp genes of S. thermophilus ST1-1, Streptococcus dysgalactiae , Streptococcus pyogenes , and Lactococcus lactis . Northern blot analysis revealed a seven- to ninefold induction of csp mRNA after a temperature shift to 20°C, showing that this thermophilic bacterium indeed contains at least one cold-inducible csp gene and that its regulation takes place at the transcriptional level.

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus pneumoniae , Enterococcus faecalis , and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac , xylA , and lacA promoters in B. subtilis and in S. pyogenes . Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.

Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium Bacillus cereus specifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of single B. cereus cells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression in B. cereus . With a case study concerning cotD expression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show that cotD is not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens . Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB , which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM , ylxF , yloA , ykoJ , and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA , htrB , and, possibly, citM . In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (σ D -dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the α-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for σ D -dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis , and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis , which have no counterparts in Lactococcus lactis , and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes ( pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene ( P nisA ). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis . Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of P nisA :: pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools.

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis . When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study ( D 120°C , 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.