An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

Use of low nutrient enrichments to access novel amylase genes in silent diversity of thermophiles

By combining low nutrient enrichments and molecular methods, a high diversity of new amylase genes was detected in a neutral sulphide-rich hot spring in Iceland. Enrichments based on hot spring water and low concentrations of starch were used to select slow-growing, starch-degrading microorganisms. Six enrichments had in total 17 bacterial types detected by 16S rRNA analysis, mostly related to the Thermus-Deinococcus group, green non-sulphur bacteria, gram positives, and uncultivated new candidate divisions. No Archaea were found. The apparent 16S rRNA species composition of the enrichments was very different from that of the microbial mat in the same hot spring. DNA samples obtained from 4 enrichments and from hot spring biomass were screened by PCR for amylase genes in glycoside-hydrolase family 13. Degenerate primers, based on conserved amino acid sequences from multiplealignments of family 13, enabled the detection of 18 amylase sequence types in the enrichments, including -amylases, -glucosidases, 1,4--glucan branching enzymes, cyclomaltodextrin hydrolases, maltogenic amylases and neopullulanases, and unspecified family 13 glycoside-hydrolases. Only one unique neopullulanase sequence, also found in most of the enrichments, was detected in the hot spring biomass DNA. The results suggest that the enrichment method combined with sequence-based screening is an efficient way to access the silent, i.e. not detectable, gene diversity in natural environments.     

Biological treatment of shrimp production wastewater

Over the last few decades, there has been an increase in consumer demand for shrimp, which has resulted in its worldwide aquaculture production. In the United States, the stringent enforcement of environmental regulations encourages shrimp farmers to develop new technologies, such as recirculating raceway systems. This is a zero-water exchange system capable of producing high-density shrimp yields. The system also produces wastewater characterized by high levels of ammonia, nitrate, nitrite, and organic carbon, which make waste management costs prohibitive. Shrimp farmers have a great need for a waste management method that is effective and economical. One such method is the sequencing batch reactor (SBR). A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same reactor to take the place of multiple reactors in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor system. This is achieved through reactor operation in sequences, which includes fill, react, settle, decant, and idle. A laboratory scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentrations of carbon and nitrogen. By operating the reactors sequentially, namely, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon. Ammonia in the waste was nitrified within 4 days. The denitrification of nitrate was achieved by the anoxic process, and 100% removal of nitrate was observed within 15 days of reactor operation.     

Increased constraints on MC4R during primate and human evolution

The melanocortin 4 receptor (MC4R) is routinely investigated for the role it plays in human obesity, as mutations in MC4R are the most common dominantly inherited form of the disease. As little is known about the evolutionary history of this locus, we investigated patterns of variation at MC4R in a worldwide sample of 1,015 humans from 51 populations, and in 8 central chimpanzees. There is a significant paucity of diversity at MC4R in humans, but not in chimpanzees. The spectrum of mutations in humans, combined with the overall low level of diversity, suggests that most (if not all) of the observed non-synonymous polymorphisms are likely to be transient deleterious mutations. The MC4R coding region was resequenced in 12 primate species and sequences from an additional 29 vertebrates were included in molecular evolutionary analyses. MC4R is highly conserved throughout vertebrate evolution, and has apparently been subject to high levels of continuous purifying selection that increased approximately threefold during primate evolution. Furthermore, the strong selection extends to codon usage bias, where most silent mutations are expected to be either quickly fixed or removed from the population, which may help explain the unusually low levels of silent polymorphisms in humans. Finally, there is a significant tendency for non-synonymous mutations that impact MC4R function to occur preferentially at sites that are identified by evolutionary analyses as being subject to very strong purifying selection. The information from this study should help inform future epidemiological investigations of MC4R. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>13</Superscript>C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a ¹³CH₃-reporter attached. This ¹³C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Estimating number of families for an urban fox population by using two public data sets

The number of families in the urban fox population of Sapporo, Japan, was estimated from two sets of data reported by the public to government: records of road-killed foxes (information-A) and records of complaints about foxes (information-B). We assumed that fox populations consist of families that have exclusive home ranges, i.e., territories, during the period between gestation and dispersal. The urban area was then divided into hexagons that correspond to the territories. The locations from the two sets of records during the territorial period were plotted on the map. The number of fox families for which information-A and/or B was reported was estimated by counting the number of hexagons that include the record. The total number of families was estimated by using a double-observation method. We adopted Chapman’s unbiased estimator which is based on the hypergeometric distribution that corresponds to the conditional likelihood. We demonstrated the possibility of estimating the abundance of animals from government data such as road kill and complaints if the animals have territories. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The High Biofilm-Encoding <Emphasis Type="Italic">Bee</Emphasis> Locus: A Second Pilus Gene Cluster in <Emphasis Type="Italic">Enterococcus</Emphasis> <Emphasis Type="Italic">faecalis</Emphasis>?

An Enterococcus faecalis mutant strain with a reduced ability for biofilm formation and primary attachment when compared to the high biofilm-forming wild-type strain was characterized by molecular biological and proteomic approaches. A point mutation in the srt-1 gene, which encodes a sortase-type enzyme and is part of the recently described bee (biofilm enhancer in Enterococcus) gene cluster, could be identified in the mutant strain. The Srt-1 deficiency resulted in a loss of the Bee-2 protein within a high molecular weight complex in cell surface protein extracts, as determined by mass spectrometry. These findings strongly suggest a specific linkage of Bee-2 to Bee-1 and Bee-3 within a complex by Srt-1. Furthermore, the identification of specific pilin motifs conserved in surface proteins of gram-positive bacteria indicated a possible involvement of the bee genes in the formation of pili structures, and may thus play a role in enhancing biofilm formation in Enterococcus faecalis.     

Mice with Disrupted TGFβ Signaling Have Normal Cerebella Development,but Exhibit Facial Dysmorphogenesis and Strain-Dependent Deficits in Their Body Wall

The transforming growth factor betas (TGFβs) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfβ1 or Tgfβ2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFβ receptors are mentally retarded, suggesting that the TGFβ isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE × PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFβ signaling. The NSE × PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFβ signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1–3 alleles, which modulate the phenotype of Tgfβ1^−/− mice, were not major determinants of the NSE × PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFβ signaling, although this may be dependent on the genetic background.