Expression of a β-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A β-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high β-galactosidase activity but utilized lactose only slightly faster than the recipient. β-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the β-galactosidase gene. Growth rates of Lac⁺ H1 and 7962 derivatives were not affected after introduction of the clostridial β-galactosidase, even though β-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-β-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-β-galactosidase activity. We suggest that β-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-β-galactosidase genes.     

A Novel GFP-Fused Eukaryotic Membrane Protein Expression System in Lactococcus lactis and Its Application to Overexpression of an Elongase

Ultraviolet (UV) irradiation has high potential to inactivate a wide range of biologic agents and is one of several nonadditive technologies being studied. The photoinactivation property of pulsed UV laser radiation (at wavelengths of 355 and 266 nm), used as an effective physical means to inactivate two typical microorganisms, prokaryotic (Escherichia coli K12) and eukaryotic (Saccharomyces cerevisiae), with respect to dose and exposure times, was examined. An E. coli population of 1.6 × 10⁴ colony-forming units (CFU)/ml was inactivated with a dose of 16.7 J/cm² energy at 355-nm wavelength. However, E. coli cells at higher concentrations were inactivated by only 98% using the same dose. Interestingly, an E. coli population of 2 × 10⁷ CFU/ml was completely inactivated using only 0.42 J/cm² at 266-nm wavelength (P ≤ 0.05). With respect to S. cerevisiae, the results were similar to those of E. coli irradiation considering that S. cerevisiae is 100 times larger than E. coli. A dose of 16.7 J/cm² completely inactivated an S. cerevisiae population of 6 × 10³ CFU/ml at 355-nm wavelength. Exposure to 266-nm wavelength, with energy doses of 1.67, 0.835, and 0.167 J/cm², successfully inactivated S. cerevisiae populations of 3 × 10⁶, 1.4 × 10⁵, and 1.5 × 10⁴ CFU/ml, respectively (P ≤ 0.05). In conclusion, compared with 355-nm wavelength, a pulsed UV laser at 266-nm wavelength inactivated a high titer of bacterial and yeast indicator standards suspended in phosphate-buffered saline-A.     

Construction and Application of a Food-Grade Expression System for Lactococcus lactis

A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000Δalr by double-crossover recombination using temperature-sensitive integration plasmid pG⁺host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry.     

Zirex: a Novel Zinc-Regulated Expression System for Lactococcus lactis

Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and P_czcD. P_czcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.     

Membrane Proteases and Aminoglycoside Antibiotic Resistance

We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products.     

Functional Expression of Bacterial Zymobacter palmae Pyruvate Decarboxylase Gene in Lactococcus lactis

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

酸适应对一株乳酸乳球菌膜脂肪酸组成和膜蛋白表达的影响

[目的]筛选具有较强酸适应能力的菌株,研究酸适应对其膜脂肪酸组成和膜蛋白表达的影响.[方法]从20株菌中筛选出一株具有较强酸适应能力的乳酸乳球菌KLDS4.0312,以GC-MS法测定该菌酸适应前后膜脂肪酸组成变化;对酸适应前后该菌膜蛋白的差异表达进行双向电泳分析.[结果]酸适应后,该菌膜不饱和脂肪酸含量从30.77%上升到42.93%,饱和脂肪酸含量从69.23%下降到57.07%,且有一种新的长链单不饱和脂肪酸C<,19:1>-n6被诱导产生.酸适应过程中至少有65个蛋白质点表达出现显著差异,其中上调的蛋白质点有43个,减弱表达的蛋白质点有22个.而添加氯霉素后,菌株的酸适应能力消除,可能与氯霉素抑制新蛋白的合成有关.[结论]说明细胞膜脂肪酸组成的适应性改变和应激蛋白的诱导产生是该菌主要的酸适应机制.     

Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.     

α-Ketoglutarate biosynthesis in wild and industrial strains of Lactococcus lactis

Aims:  This study was carried out to explore the ability of wild and industrial strains of Lactococcus lactis to produce α-ketoglutarate (α-KG), which is essential during the conversion of amino acids to flavour compounds.
Methods and Results:  Two pathways in α-KG biosynthesis were explored in strains of L. lactis isolated from dairy products, vegetables and commercial dairy starter cultures. Half of the strains efficiently converted glutamine to glutamate (Glu) and grew in Glu-free medium. Strains did not present isocitrate dehydrogenase and aconitase activities. However, half of the strains presented glutamate dehydrogenase (GDH) activity.
Conclusions:  The ability of L. lactis to synthesize either α-KG or Glu via GDH was confirmed. However, L. lactis strains were not able to biosynthesize α-KG by the citrate–isocitrate pathway. NADP-GDH activity was mainly found in strains isolated from vegetables, whereas NAD-GDH activity was mainly found in strains isolated from dairy products.
Significance and Importance of the Study:  The origin of isolation highly influenced NAD or NADP-GDH activities. These enzymatic activities may be correlated to the flavour production capacity of the different strains.     

An Alternative Gelling Agent for Culture and Studies of Nematodes,Bacteria, Fungi,and Plant Tissues

Pluronic F127 polyol, a block copolymer of propylene oxide and ethylene oxide, was studied as an alternative to agar in culture media for nematodes, bacteria, fungi, actinomycetes, and plant tissues or seedlings, At a polyol concentration of 20% w/v, the culture media, semi-solid at room temperature (22 C) but liquid at lower temperatures, had minimal effects on the test organisms. Most of the fungi and bacteria grew as well in 20% polyol as in 1.5% agar media; however, various species of nematodes and plant seedlings or tissues exhibited differential sensitivities to different concentrations of the polyol. In cases where the organisms were unaffected, the polyol media had certain advantages over agar, including greater transparency and less contamination under nonaseptic conditions. Polyol media have potentially greater ease for recovery of embedded organisms or tissues inside the media by merely shifting to lower temperatures.     

Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606^T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.     

Reconstruction of the proteolytic pathway for use of β-casein by Lactococcus lactis

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L . lactis liberates the required amino acids from β-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo . To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of β-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo  ; (iii) use of oligopeptides of 5–10 residues becomes only possible after uptake via Opp; (iv) only a few (10–14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.     

Functional Analysis of Glycosyltransferase Genes from Lactococcus lactis and Other Gram-Positive Cocci: Complementation, Expression, and Diversity

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.