Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway

The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (KM=0.014mM) and maximum rate (Vmax=11.2μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.

Increasing incidence of Clostridium difficile infection, Australia, 2011–2012

Objectives: To report the quarterly incidence of hospital-identified Clostridium difficile infection (HI-CDI) in Australia, and to estimate the burden ascribed to hospital-associated (HA) and community-associated (CA) infections. Design, setting and patients: Prospective surveillance of all cases of CDI diagnosed in hospital patients from 1 January 2011 to 31 December 2012 in 450 public hospitals in all Australian states and the Australian Capital Territory. All patients admitted to inpatient wards or units in acute public hospitals, including psychiatry, rehabilitation and aged care, were included, as well as those attending emergency departments and outpatient clinics. Main outcome measures: Incidence of HI-CDI (primary outcome); proportion and incidence of HA-CDI and CA-CDI (secondary outcomes). Results: The annual incidence of HI-CDI increased from 3.25/10 000 patient-days (PD) in 2011 to 4.03/10 000 PD in 2012. Poisson regression modelling demonstrated a 29% increase (95% CI, 25% to 34%) per quarter between April and December 2011, with a peak of 4.49/10 000 PD in the October–December quarter. The incidence plateaued in January–March 2012 and then declined by 8% (95% CI, − 11% to − 5%) per quarter to 3.76/10 000 PD in July–September 2012, after which the rate rose again by 11% (95% CI, 4% to 19%) per quarter to 4.09/10 000 PD in October–December 2012. Trends were similar for HA-CDI and CA-CDI. A subgroup analysis determined that 26% of cases were CA-CDI. Conclusions: A significant increase in both HA-CDI and CA-CDI identified through hospital surveillance occurred in Australia during 2011–2012. Studies are required to further characterise the epidemiology of CDI in Australia.

Epidemiology of Clostridium difficile infection (CDI) in Queensland, Australia

Background: International epidemic clones (ribotypes 027 and 078) of Clostridium difficile have been associated with death, toxic megacolon and other adverse outcomes in North America and Europe. In 2010, the first local transmission of an epidemic strain (027) of C. difficile was reported in the state of Victoria, Australia, but no cases of infection with this strain were reported in the state of Queensland. In 2012, a prevalence study was undertaken in all public and selected private hospitals to examine the epidemiology of CDI and determine the prevalence of epidemic C. difficile strains in Queensland. Methods: Enhanced surveillance was undertaken on all hospital identified CDI cases aged over 2 years between 10 April and 15 June 2012. Where available, patient samples were cultured and isolates of C. difficile ribotyped. The toxin profile of each isolate was determined by PCR. Results: In total, 168 cases of CDI were identified during the study period. A majority (58.3%) of cases had onset of symptoms in hospital. Of the 62 patients with community onset of symptoms, most (74%) had a hospital admission in the previous 3 months. Only 4 of 168 patients had onset of symptoms within a residential care facility. Thirteen out of the 168 (7.7%) patients included in the study had severe disease (ICU admission and/or death within 30 days of onset). Overall 136/168 (81%) of cases had been prescribed antibiotics in the last month. Of concern was the emergence of a novel ribotype (244) which has recently been described in other parts of Australia and is genetically related to ribotype 027. Seven patients were infected with C. difficile ribotype 244 (8% of 83 samples ribotyped), including one patient requiring ICU admission and one patient who died. Ribotype 244 was tcdA, tcdB and CDT positive and contained a tcdC mutation at position 117. Conclusion: Ongoing surveillance is required to determine the origin and epidemiology of C. difficile ribotype 244 infections in Australia.

Leptospirosis and Leptospires - The silent assassins

Leptospirosis is one of the most common yet under reported zoonoses. Leptospires, the etiological agents of leptospirosis are ubiquitous pathogens, with a world-wide distribution, causing a spectrum of disease ranging from a mild influenza-like illness to Weil’s disease, which manifests itself in multi-organ failure. The following chapter reports on the epidemiology and transmission of the disease in humans and animals. The chapter will also delineate the symptoms observed in humans and animals and in concluding outline unresolved and evolving issues for microbiologists, epidemiologists and public health officials.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Methods for isolation and cultivation of filamentous fungi

Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology.

Enzyme activities and biotechnological applications of cold-active microfungi

Fungi are eukaryotic organisms and considered to be less adaptable to extreme environments when compared to bacteria. While there are no thermophilic microfungi in a strict sense, some fungi have adapted to life in the cold. Cold-active microfungi have been isolated from the Antarctic and their enzyme activities explored with a view to finding new candidates for industrial use. On another front, environmental pollution by petroleum products in the Antarctic has led to a search for, and the subsequent discovery of, fungal isolates capable of degrading hydrocarbons. The work has paved the way to developing a bioremedial approach to containing this type of contamination in cold climates. Here we discuss our efforts to map the capability of Antarctic microfungi to degrade oil and also introduce a novel cold-active fungal lipase enzyme.

The knowledge base of subject-matter experts in teaching : A case study of a professional scientist as a beginning teacher

One method of addressing the shortage of science and mathematics teachers is to train scientists and other science-related professionals to become teachers. Advocates argue that as discipline experts these career changers can relate the subject matter knowledge to various contexts and applications in teaching. In this paper, through interviews and classroom observations with a former scientist and her students, we examine how one career changer used her expertise in microbiology to teach microscopy. These data provided the basis for a description of the teacher’s instruction which was then analysed for components of domain knowledge for teaching. Consistent with the literature, the findings revealed that this career changer needed to develop her pedagogical knowledge. However, an interesting finding was that the teacher’s subject matter as a science teacher differed substantively from her knowledge as a scientist. This finding challenges the assumption that subject matter is readily transferable across professions and provides insight into how to better prepare and support career changers to transition from scientist to science teacher.

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and micro-biological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Statistical analysis of reduction in tensile strength of cotton strips as a measure of soil microbial activity

The cotton strip assay (CSA) is an established technique for measuring soil microbial activity. The technique involves burying cotton strips and measuring their tensile strength after a certain time. This gives a measure of the rotting rate, R, of the cotton strips. R is then a measure of soil microbial activity. This paper examines properties of the technique and indicates how the assay can be optimised. Humidity conditioning of the cotton strips before measuring their tensile strength reduced the within and between day variance and enabled the distribution of the tensile strength measurements to approximate normality. The test data came from a three-way factorial experiment (two soils, two temperatures, three moisture levels). The cotton strips were buried in the soil for intervals of time ranging up to 6 weeks. This enabled the rate of loss of cotton tensile strength with time to be studied under a range of conditions. An inverse cubic model accounted for greater than 90% of the total variation within each treatment combination. This offers support for summarising the decomposition process by a single parameter R. The approximate variance of the decomposition rate was estimated from a function incorporating the variance of tensile strength and the differential of the function for the rate of decomposition, R, with respect to tensile strength. This variance function has a minimum when the measured strength is approximately 2/3 that of the original strength. The estimates of R are almost unbiased and relatively robust against the cotton strips being left in the soil for more or less than the optimal time. We conclude that the rotting rate X should be measured using the inverse cubic equation, and that the cotton strips should be left in the soil until their strength has been reduced to about 2/3.

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism

The SOS screen, as originally described by Perkins et al. (1999), was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS+ candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS+ candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS+ candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.