Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex

Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content

The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R. N. Brey and B. P. Rosen, J. Bacteriol. 139:824-834, 1979). In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias. Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays. The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis. Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM. The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively. Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming). Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate). The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis. omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E. coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit

The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia

Background: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb2+, Al+3, SeO2, Cu2+, AsO4, and Zn2+. With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. Results: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. Conclusions: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils

Influence of Temperature on the Physiology and Virulence of the Insect Pathogen Serratia sp. Strain SCBI

The physiology of a newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), which is both a nematode mutualist and an insect pathogen, was investigated and compared to that of Serratia marcescens Db11, a broad-host-range pathogen. The two Serratia strains had comparable levels of virulence for Manduca sexta and similar cytotoxic activity patterns, but motility and lipase and hemolytic activities differed significantly between them

Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein

R-factor mediated bacterial resistance to arsenical salts occurs by active extrusion of the toxic oxyanions from cells of gram negative bacteria. The ars operon of the conjugative plasmid R773 encodes an anion pump. The pump has two polypeptide components. The catalytic subunit, the ArsA protein, is an oxyanion-stimulated ATPase. The membrane component, the ArsB protein, has been localized in the inner membrane of Escherichia coli. The ArsA and ArsB proteins have been postulated to form a membrane complex which functions as an anion-translocating ATPase. In this study evidence is presented showing that expression of the arsB gene is required to anchor the ArsA protein to the inner membrane. Binding studies with purified ArsA to membranes with and without the arsB gene product confirm this requirement. Membranes of uncA mutants containing both the ArsA and ArsB proteins exhibit arsenite(antimonite)-stimulated ATPase activity. These results support the model in which the ArsA protein is the catalytic energy transducing component of the anion pump, whereas the integral membrane ArsB protein serves as both the anion channel and membrane binding site for the ArsA protein

Preservice Teachers’ Creation Of And Beliefs About Culturally Responsive And Critical Mathematics Tasks, And Their Beliefs About The Students For Whom Their Tasks Were Intended

In this study, case study methodology was used to explore secondary mathematics preservice teachers (PSTs) attempts to construct tasks for lower income, predominantly African American students at a cultural immersion site who PSTs encountered frequently and interviewed. PST beliefs about their tasks and their students were also investigated. The first research aim of this study was to investigate PSTs’ attempts to create culturally responsive mathematics tasks for themselves and for the students. PSTs incorporated students’ personal and cultural details in tasks for the students. However, they had greater difficulty integrating cultural backdrops into the primary mathematics activities of tasks they created for their students than integrating cultural backdrops into tasks they created for themselves. Additionally, they distorted students’ cultural contexts by filtering student details through their own lenses. The second research aim was to investigate PSTs’ attempts to create critical mathematics tasks for the students, as well as PSTs’ beliefs about the tasks they created. Most PSTs identified a social justice issue in their tasks, but did not require students to question the fairness of the disparate treatment of the disenfranchised. Also, most tasks explicitly related to social class issues. Despite expressing critical consciousness and agency benefits, PSTs believed that their tasks were inappropriate for middle school-aged students because of the tasks’ “uncomfortable” or “controversial” nature. The study’s final aim was to investigate micro-transformations, or shifts, in PSTs’ beliefs about the students. All of the four PSTs chosen for this part of the examination experienced micro-transformations in their beliefs about the students. PSTs with past encounters with lower income or minority students experienced micro-transformations earlier than other PSTs. The study identified a need for professional development that allows PSTs to learn about cultural others and disenfranchised people from culturally different and disenfranchised students, critical educators, and critical friends, and a need for instrument development that assesses PST tasks and determines appropriate support for PSTs in the construction of tasks for culturally different and disenfranchised students

Comparative Study on Criminal Provisions on Regional Regulations Concerning Exclusive Breastfeeding

Infant’s right on exclusive breastfeeding has been protected on Law No. 36/2009 on Health. One of the measurements in its protection is by using criminal provision. It is stated in Art. 200 and 201, each of which has criminal sanction to those obstructing the exclusive breastfeeding program. As an effort to implement this national policy, quite a numerous of provinces or even lower administrative regions had issued regulations concerning the same matter. Some of them have criminal provision as well, as can be seen in City Regulation of Cirebon No. 4/2016 on Early Initiation and Exclusive Breastfeeding and also in the City Regulation of Semarang No. 5/2014 on Early Initiation and Exclusive Breastfeeding. This paper will assess specifically the criminal provisions on those regional laws in a comparative perspective. The selection of the two laws is based on the preliminary study which found a quite unique form of criminal provisions on each laws. The analysis is also be based on art 200 and 201 Law No. 36/2009 and its derivative regulations as a normative measurement in national level, with which the two regional laws must be in line to. This research found that there are quite a significance differences between the two laws especially regarding the form of action that is criminally regulated. Variation also found on how the two laws fulfil what is demanded by the national criminal policy

Calcium Homeostasis in Escherichia coli: Characterization of Mutants and Genome Expression of MG1655

While the role of calcium ions as secondary messengers has been well described in eukaryotic cells, little is known about calcium homeostasis in bacteria at the physiological and molecular levels. Genetic and genomic approaches were used to address calcium regulation and to identify genes (cal) involved in calcium homeostasis. Transposon mutagenesis of Escherichia coli generated several calcium-sensitive mutants that fell into three categories: (i) Ca2+-sensitive chemotaxis mutants, (ii) Ca2+-sensitive cell division mutants, and (iii) Ca2+-sensitive mutants that showed no defects in cell division or chemotaxis. The physiological properties of these Ca2+-sensitive mutants were determined. Besides calcium-sensitivity to 75 mM calcium, all of the mutants exhibited increased sensitivities to several divalent cations including Ni2+, Mg2+, Mn2+, Co2+, Zn2+, Cu2+, and Cd2+. To identify the cal gene sequence in the Ca2+-sensitive mutants, the region of the genes fused to the reporter gene (phoA) on the transposon TnphoA was amplified by PCR and sequenced. The sites of the gene fusion for three cal mutants were at the fdoG, gpt and pqi5 genes. The pleiotropic nature for the cal mutations suggested that many genes may be globally regulated by calcium. We then investigated global gene expression patterns of wild-type E. coli under calcium-depleted (addition of 10 mM EGTA) and calcium-elevated (addition of 75 mM Ca2+) conditions as compared to cultures grown under unstressed conditions. A comprehensive transcriptome analysis using macroarrys exhibited a global regulation of diverse genes within the E. coli genome during calcium homeostasis

PENGARUH OBJEK WISATA DANAU SEBEDANG TERHADAP KONDISI SOSIAL EKONOMI MASYARAKAT KECAMATAN SEBAWI KABUPATEN SAMBAS

AbstractThis study aims to determine the effect of Lake Sebedang tourism objects on the socioeconomic conditions of the people of Sebawi District, Sambas Regency. The data analysis used in this research is simple regression analysis with the form of quantitative research. The population in this study were the people of Sebawi District, Sambas Regency who live around the Sebedang Lake tourism object with a total sample of 52 respondents. The data collection technique used is indirect communication techniques. Based on a simple regression analysis, the research results show the equation Y = 14.041 + 0.651 X which means a constant value of 14.041, i.e. if a tourist attraction (X) is 0 then the socio-economic condition (Y) is 14.041. This means that every increase in tourist objects (X) by 1, the socioeconomic conditions (Y) will increase by 0.651. The magnitude of the influence of variable X on variable Y (coefficient of determination) is 0.292, which means that the percentage of Lake Sebedang tourism objects on the socio-economic conditions is 29.2%, while the rest is influenced by other factors.Keywords: Lake Sebedang Socio-Economic Conditions, Tourist Attraction