Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Thermal tolerance of potential <Emphasis Type="Italic">Trichogramma</Emphasis> strains for mass-production and paddy field release in the Greater Mekong Subregion

In order to identify suitable Trichogramma strains for mass production and successful control of key lepidopteran pests in paddy fields in the Greater Mekong Subregion, the effects of high temperatures on wasp life history traits were compared among three strains of Trichogramma chilonis Ishii and two strains of Trichogramma ostriniae Pang et Chen, collected from paddy fields in the region. At a rearing temperature of 25 °C, life history traits differed significantly among the five strains tested and the three T. chilonis strains and a T. ostriniae strain were of high performance. When female wasps were exposed to higher temperatures commonly encountered in the region, negative effects were observed on key life history parameters of adult females and their offspring at 34 °C, which became even more serious at 37 °C. In particular, the two T. ostriniae strains were not able to successfully develop to adulthood at all at 37 °C while for the T. chilonis strains adult emergence was significantly reduced. In addition, the emerged offspring females lived only around one day and no parasitism was observed. When the three T. chilonis strains were exposed to 37 °C for 4 to 12 h at prepupal and pupal stages, reflecting heat shocks that the released Trichogramma may experience in the field, adult emergence was significantly reduced after an exposure time of 12 h. In summary, the three T. chilonis strains show a relatively high potential for incorporation in a biological control program in the target region. Our results also highlight that tests at both rearing and field temperatures are necessary in selection of potential Trichogramma strains for an inundative release program where there is significant difference between rearing and field temperatures.     

Diversity and enzymatic potentialities of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strains isolated from a polluted freshwater ecosystem in Cuba

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

Selection and Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Berliner) (Eubacteriales: Bacillaceae) Strains for <Emphasis Type="Italic">Ecdytolopha aurantiana</Emphasis> (Lima) (Lepidoptera: Tortricidae) Control

The citrus fruit borer, Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), is responsible for major losses to the citrus industry because it causes rot and drop of fruits. The current study aimed to select and characterize Bacillus thuringiensis (Berliner, 1911) strains toxic to E. aurantiana. For this purpose, 47 B. thuringiensis strains were evaluated in selective bioassays using first instar larvae of E. aurantiana. The lethal concentration (LC₅₀) of the most toxic strains was estimated, and the strains were characterized by morphological, biochemical, and molecular methods. Of the 47 strains tested, 10 caused mortality above 85% and showed mean lethal concentrations between 1.05E+7 and 1.54E+8 spores mL^?1. The lowest LC₅₀ values were obtained for the HD-1 standard strain and the BR145, BR83, BR52, and BR09 strains. The protein profile showed the presence of Cry proteins of 60, 65, 70, 80, and 130 kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes. The morphological analysis identified three different crystalline inclusions: bipyramidal, round, and cuboidal. The cry1 and cry2 genes were the most frequent among the B. thuringiensis strains evaluated and encode Cry proteins toxic to insects of the order Lepidoptera, which agree with the toxicity results obtained by the selective bioassays against E. aurantiana. The results showed four different B. thuringiensis strains toxic to E. aurantiana at the same level as the HD-1 standard strain, and these strains have biotechnological potential for E. aurantiana control through the production of transgenic plants or the formulation of biopesticides.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

Polymorphism of lactose genes in the dairy yeasts <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>, potential probiotic microorganisms

Molecular karyotyping and Southern blot hybridization were used to investigate chromosomal polymorphism of the LAC genes controlling lactose fermentation in Kluyveromyces marxianus strains isolated from various dairy products and natural sources in Russia and CIS countries. Profound polymorphism of karyotype patterns and accumulation of LAC genes were observed in dairy K. marxianus strains. K. marxianus strains isolated from dairy products intensively fermented lactose at 37°C after one day of cultivation, while non-dairy strains exhibited delayed lactose fermentation or did not ferment it at all. Based on the fermentation tests, twelve K. marxianus strains were selected, which are of interest as potential probiotic microorganisms suitable for further molecular genetic studies and breeding.     

In vitro assessment of safety and probiotic potential characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from water buffalo mozzarella cheese

The aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).     

Toxin Gene Contents and Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains Against Two Sugarcane Borer Species, <Emphasis Type="Italic">Diatraea saccharalis</Emphasis> (F.) and <Emphasis Type="Italic">D. flavipennella</Emphasis> (Box)

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC₅₀ values of strains for D. saccharalis varied from 0.08 × 10⁵ (LIIT-0105) to 4104 × 10⁵ (LIIT-2707) spores + crystals mL^?1. For D. flavipennella, the LC₅₀ values of strains varied from 0.40 × 10⁵ (LIIT-2707) to 542 × 10⁵ (LIIT-2109) spores + crystals mL^?1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.     

A metagenomic assessment of the bacteria associated with <Emphasis Type="Italic">Lucilia sericata</Emphasis> and <Emphasis Type="Italic">Lucilia cuprina</Emphasis> (<Emphasis Type="Italic">Diptera: Calliphoridae</Emphasis>)

Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) is a blow fly genus of forensic, medical, veterinary, and agricultural importance. This genus is also famous because of its beneficial uses in maggot debridement therapy (MDT). Although the genus is of considerable economic importance, our knowledge about microbes associated with these flies and how these bacteria are horizontally and trans-generationally transmitted is limited. In this study, we characterized bacteria associated with different life stages of Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann) and in the salivary gland of L. sericata by using 16S rDNA 454 pyrosequencing. Bacteria associated with the salivary gland of L. sericata were also characterized using light and transmission electron microscopy (TEM). Results from this study suggest that the majority of bacteria associated with these flies belong to phyla Proteobacteria, Firmicutes, and Bacteroidetes, and most bacteria are maintained intragenerationally, with a considerable degree of turnover from generation to generation. In both species, second-generation eggs exhibited the highest bacterial phylum diversity (20 % genetic distance) than other life stages. The Lucilia sister species shared the majority of their classified genera. Of the shared bacterial genera, Providencia, Ignatzschineria, Lactobacillus, Lactococcus, Vagococcus, Morganella, and Myroides were present at relatively high abundances. Lactobacillus, Proteus, Diaphorobacter, and Morganella were the dominant bacterial genera associated with a survey of the salivary gland of L. sericata. TEM analysis showed a sparse distribution of both Gram-positive and Gram-negative bacteria in the salivary gland of L. sericata. There was more evidence for horizontal transmission of bacteria than there was for trans-generational inheritance. Several pathogenic genera were either amplified or reduced by the larval feeding on decomposing liver as a resource. Overall, this study provides information on bacterial communities associated with different life stages of Lucilia and their horizontal and trans-generational transmission, which may help in the development of better vector-borne disease management and MDT methods.     

Correlations between the ages of<Emphasis Type="Italic">Alnus</Emphasis> host species and the genetic diversity of associated endosymbiotic<Emphasis Type="Italic">Frankia</Emphasis> strains from nodules

Nodule samples were collected from four alder species:Alnus nepalensis, A. sibirica, A. tinctoria andA. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. A total of 21 restriction patterns were obtained. TheFrankia population in the nodules ofA. nepalensis had the highest genetic diversity among all fourFrankia populations; by contrast, the population in the nodules ofA. mandshurica had the lowest degree of divergence; the ones in the nodules ofA. sibirica andA. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated thatFrankia strains fromA. sibirica andA. tinctoria had a close genetic relationship.Frankia strains fromA. nepalensis might be the ancestor ofFrankia strains infecting otherAlnus species. From these results and the inference of the ages ofAlnus host species, it is deduced that there was a co-evolution betweenAlnus and its microsymbiontFrankia in China.     

Detection of virulence-related genes in <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> and their expression in response to different conditions

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.     

Adherent/invasive <Emphasis Type="Italic">Escherichia coli</Emphasis> (AIEC) isolates from asymptomatic people: new <Emphasis Type="Italic">E. coli</Emphasis> ST131 O25:H4/H30-Rx virotypes

Background

The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried.

Methods

Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTX^R ST131 O25:H4/H30-Rx strains collected from healthy donors’ stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated.

Findings

Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples.

Interpretation

The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.

     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

Occurrence and characterisation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">diarizonae</Emphasis> serovar 61: k: 1, 5, (7) in sheep in the federal state of Thuringia,Germany

Background

The occurrence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) and other Salmonella organisms in sheep in the German federal state of Thuringia was examined for the first time. Pooled faecal samples from 90 flocks located in this state were monitored.

Results

Only SASd was detected in 74 (82.2%) out of the 90 sheep herds, other Salmonella serovars were not identified. A positive correlation was found between the flock size and the detection probability of SASd. Despite the agent’s high prevalence, clinical symptoms of a disease exclusively due to SASd have not been observed. The SASd strains were characterised by macrorestriction analysis, antimicrobial testing and the biochemical profile. All strains were sensitive to 13 out of 14 antimicrobial substances and resistant to only sulfamethoxazole. The high number of macrorestriction groups of SASd strains indicated a low clonality of the serovar.

Conclusions

Data from sheep derived foods and public health data in Germany strongly suggest that the significance of SASd for public health is considerably lower than that of serovars belonging to Salmonella enterica subspecies enterica. For this reason and because of the low disease-causing potential of SASd in sheep, it is worthwile to consider a reduction in ongoing activities from combating to monitoring serovar 61: k: 1, 5, (7) in the sheep population.

     

Benzalkonium chloride tolerance of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> strains isolated from a meat processing facility is related to presence of plasmid-borne <Emphasis Type="Italic">bcr</Emphasis>ABC cassette

Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L. monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes. However, the bcrABC cassette was present in 17 isolates of serogroup IIa possessing the same AscI/ApaI pulsotype, the operon being localized on a plasmid. The significant relation of BAC tolerance with bcrABC presence was confirmed as all bcrABC positive isolates showed the highest minimal inhibitory concentration (MIC) values for BAC and increased sensitivity to BAC was observed after plasmid curing. No effect of the efflux pump inhibitor reserpine on BAC tolerance in bcrABC positive strains was observed in contrast to all bcrABC negative strains. Lower ethidium bromide efflux in bcrABC positive isolates compared to bcrABC negative and plasmid-cured L. monocytogenes isolates was observed. The expression of bcrABC genes was BAC-induced. The confirmed effect of bcrABC to increased BAC tolerance, coupled with its plasmid location, may be an important factor in potential dissemination of the biocide resistance among Listeria species. The understanding of molecular mechanisms of biocide tolerance should help to improve control measures to prevent further spread of L. monocytogenes in food production environments with frequent use of BAC.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.