GENETIC CONTROL OF CYTODIFFERENTIATION

The cells of the anterior region of the larval fatbody of Drosophila melanogaster accumulate kynurenine at the end of the third larval instar, whereas the cells of the posterior region are involved in pteridine metabolism. Through a series of transplantation experiments it has been demonstrated that the anterior fat cells synthesize kynurenine. The mutant vermilion lacks kynurenine, and the anterior fat cells of this mutant strain lack the autofluorescence characteristic of kynurenine. When the non-allelic suppressor gene is combined with vermilion, the synthesis of kynurenine is restored in the anterior fat cells, and some of the cells of the posterior region contain kynurenine as well. A similar extension in the number of cells containing kynurenine can be induced in the normal Ore-R strain by feeding the precursor tryptophan. It has been concluded that the absence of a physiological process in a differentiated cell does not necessarily represent a loss of the genetic potential for that process. The normal allele at the suppressor locus inhibits the occurrence of kynurenine in the posterior fat cells, whereas the mutant allele su²-s allows the expression of this potential. An inducer such as tryptophan can overcome this inhibition in the normal strain, and as a result the cells which are normally differentiated as "isoxanthopterin cells" may produce kynurenine as well.     

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc²155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc²155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc²155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.     

Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and ¹³C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.     

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice

The NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these ??humanized mice?? (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.     

Suppression of ERK activation in urethral epithelial cells infected with Neisseria gonorrhoeae and its isogenic minD mutant contributes to anti-apoptosis

In gonococci-infected transduced human urethral epithelial cells (THUEC), the role of ERK, a mitogen-activated protein kinase (MAPK), in apoptosis is unknown. We observed lowering of ERK activation in THUEC following infection with anti-apoptosis-inducing Neisseria gonorrhoeae strain CH811. An isogenic cell division mutant of this strain, Ng CJSD1 (minD deficient), which is large and abnormally shaped, reduced ERK phosphorylation levels even more than its parental strain in THUEC. This led to higher anti-apoptosis in mutant-infected cells as compared to the parental strain-infected cells. Our results suggest that N. gonorrhoeae infection reduces ERK activation in THUEC contributing to anti-apoptosis.     

Oxidation of Methyl tert-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n-Octane-Grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K_s value (20 to 40 mM) for MTBE.     

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.     

Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens

An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC₅₀ value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.     

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.     

Properties of a new strain of Nitrosomonas isolated from an aerobic biofilm in a domestic sewage-treatment system

A new ammonia-oxidizing strain, isolated from an aerobic biofilm in a domestic sewage-treatment system, was identified as a species of Nitrosomonas different from Nitrosomonas europaea. This strain had morphological features and growth characteristics typical of members of the genus Nitrosomonas. The G+C content of the DNA of this strain was 48.5 mol%, being lower than that of known strains of N. europaea. The extent of the homology between the DNA of this strain and that of other strains of N. europaea was less than 30%. After cells of this isolate, immobilized in a polyacrylamide gel, had been added to the aerobic reactor of a laboratory-scale sewage-treatment system, the concentration of ammonium nitrogen in the effluent decreased to 2 mg/l without the accumulation of nitrite, and removal of more than 70% of the nitrogen from the input sewage was achieved.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Biological properties of the wild rhizosphere strain Pseudomonas fluorescens 2137 and its derivatives marked with the gusA gene

he natural wild rhizosphere strain P. fluorescens 2137 was marked with the β-glucuronidase gene gusA. The introduction of this gene influenced the viability of the wild strain, as well as its certain physiological parameters, such as cultural characteristics, biochemical properties, and antagonistic activity against the phytopathogenic fungi Fusarium culmorum, F. oxysporum, F. graminearum, and Verticillum nigrescens. The gusA-marked derivative strains that deviate the least from the wild strain in biological properties can be used to monitor populations of P. fluorescens 2137 cells in the plant rhizosphere.     

A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.     

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.     

Transformation of Acinetobacter calco-aceticus (Bacterium anitratum)

A highly efficient transformation system has been demonstrated in a strain of Acinetobacter calco-aceticus (Bacterium anitratrum). During mixed growth of various stable, unencapsulated, mutant strains, deoxyribonucleic acid (DNA) is liberated and fully encapuslated transformants can be isolated. Purified DNA preparations have been used to transform suitable recipient mutant strains for ability to synthesize capsules, ability to dispense with a growth factor requirement, and resistance to streptomycin. When the wild-type strain is deprived of its capsule, either by mechanical stripping or by mutation, the unencapsulated cells tend to form large clumped masses. A nonclumping mutant of an unencapsulated strain has been isolated. When ability to synthesize capsules is transformed into this nonclumping strain, the resultant cells no longer form chains, unlike the wild-type encapsulated strain. It appears likely that the occurrence of transformation during growth of mixed cultures, with glucose or gluconate as the carbon source, may be the result of osmotic rupture resulting from the inability of unencapsulated strains to oxidize triose phosphates as fast as they are formed. The finding of transformation in Acinetobacter may provide an additional useful organism for the study of this mode of genetic transfer since this strain grows well in a simple mineral medium containing a single oxidizable source of carbon. Furthermore, no special supplementary factors seem to be required for transformation to take place.     

Enhanced hydantoin-hydrolyzing enzyme activity in an Agrobacterium tumefaciens strain with two distinct N-carbamoylases

Hydantoin-hydrolyzing enzymes of Agrobacterium tumefaciens isolates such as strain RU-OR are used as biocatalysts in the commercial production of d-hydroxyphenylglycine via hydrolysis of d, l-p-hydroxyphenylhydantoin. Hydantoin-hydrolyzing enzyme activity in RU-OR cells is tightly regulated by nitrogen catabolite repression and is induced when hydantoin or a hydantoin-analogue is present in the growth medium. Previous studies have selected mutant strains which are inducer-independent and no longer subject to nitrogen catabolite expression. However, these mutants did not exhibit significantly higher levels of enzyme activity compared to the wild-type strain. In this study, we have focused on enhancing the levels of hydantoinase and N-carbamoylase activity in wild-type RU-OR cells by manipulating the growth medium or over-expressing the global nitrogen regulatory factors, NtrBC. We also show that this strain encodes two distinct d-selective N-carbamoylases. One enzyme is virtually identical to the other Agrobacterium N-carbamoylases while the second represents a new class of d-N-carbamoylases with potentially novel biocatalytic properties.     

Probiotic Activity of a Bacterial Strain Isolated from Ancient Permafrost Against Salmonella Infection in Mice

Bacillus cereus strain F, collected from relict permafrost located in Siberia, was analyzed for probiotic activity in the mouse Salmonella enterica model. Viable bacterial cells were found in frozen soils taken at Mammoth Mountain in Yakutia from a depth below the level of seasonal thawing. Geological data indicated the absence of a thawing within millions of years of deposited soils, which helped to ensure the ancient origin of our sample. According to DNA analysis, bacterial cells collected from the relict permafrost appeared to be B. cereus strain F. The morphology of these bacteria was analyzed using atomic force microscopy. B. cereus strain F was assessed as a nonpathogenic bacterium by evaluation of its pathogenicity. A S. enterica model is described in mice after per oral inoculation and serves as a model for the human carrier state. Using this model, probiotic activity by the bacterial strain isolated from the ancient permafrost has been shown against Salmonella infection in mice.