Cytoskeleton proteins involved in chromosome segregation and cell division in Corynebacterium glutamicum

The chromosome partitioning system of the rod-shaped actinomycete, Corynebacterium glutamicum consists of the Walker-type ATPase (ParA), a DNA binding protein (ParB) and centromere-like parS sites, located at the origin-proximal region of the chromosome. ParB binds parS sites, specifically. ParA is recruited to the ParB-parS nucleoprotein complex, likely providing the driving force needed to relocalize replicated oriC’s to the opposite cell pole. The ParB-oriC complex is then stably attached to the cell pole, where it remains and the cell divides in-between the segregated chromosomes. The phenotypic consequences of mutation of parA or parB include reduced growth rates, a high frequency of anucleate cells and altered cell lengths. Also, in the absence of parA, the oriC is mislocalized. To date, polar origin tethering factors have been identified in only few bacteria. Thus, we wanted to identify and analyze the Actinobacteria chromosome polar targeting factor. A synthetic in vivo approach was employed to analyze the anchoring of the ParB-oriC nucleoprotein complex to the cell poles via interaction with DivIVA. It was shown that DivIVA is necessary and sufficient to recruit ParB, therefore also tether the oriC at the cell poles. With this synthetic system, in combination with mutational analysis, the interaction sites between ParB and DivIVA were mapped. In Corynebacterium glutamicum, mutation of the N-terminal of ParB showed reduced polar oriC localization. In addition, the interaction between ParB and DivIVA was demonstrated for other members of the Actinobacteria phylum, including the notorious pathogen Mycobacterium tuberculosis and Streptomyces coelicolor. Corynebacterium glutamicum, which undergoes cell division between the segregated nucleoids but not necessarily precisely at midcell, does not possesses the conventional positive or negative FtsZ regulators found in other rod-shaped bacteria. However, Corynebacterium glutamicum encodes an orphan parA-like gene (pldP, for ParA-like Division Protein). In this thesis a number of subtle differences between ParA and PldP were highlighted, showing that PldP is not involved in chromosome segregation, but probably in regulation of cytokinesis. Similar to the MinD protein of B. subtilis, PldP contains a putative C-terminal amphipathic helix. In vivo, PldP localizes, probably early, to the division septum and the localization pattern is highly reminiscent of MinD. Further in vitro analysis showed that PldP can bind membranes. Contrary to the long-standing assumption, some Corynebacterium glutamicum strains encode an actin homologue. The cg1890 (designated AlpC, for Actin-Like Protein Corynebacterium) gene was recently identified as a putative actin-like protein. At the sequence level, AlpC shares little homology with actin and other actin-like proteins, however, it does contain the actin signature motive involved in nucleotide binding and hydrolysis. In vivo, AlpC forms dynamic structures, which assemble into long straight filaments and dissemble into foci. In vitro, AlpC can hydrolyze both ATP and GTP and exhibits nucleotide dependent polymerization. Mutation of the actin signature motif (AlpCD301A) abolishes nucleotide hydrolysis but not polymerization. Thus, AlpC is a genuine member of the actin-like superfamily. On the genome, alpC is one of the first on the CGP3 prophage region. This prophage no longer forms infectious phage particles and most of the coding regions show little homology to known bacterial genes. The CGP3 prophage can excise from the bacterial chromosome and exist as multiple copies of circular DNA. In vivo co-visualization of induced prophage and AlpC filaments suggests that the Corynebacterium glutamicum actin-like protein is not involved in segregation of the prophage particles. However, in the absence of alpC, the frequency of prophage induction is drastically reduced. Thus, we speculate that AlpC plays a role in replication the CGP3 prophage

Chromosome segregation impacts on cell growth and division site selection in Corynebacterium glutamicum.

Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids

‘White excellence and black failure’: the reproduction of racialised higher education in everyday talk

Since the advent of democracy in South Africa in 1994, much effort has been expended on overcoming the institutionalised racism that characterised apartheid. The transformation of higher education, particularly with regard to the merging and incorporation of institutions, is such an example. This article is an analysis of discourses on race emerging in the talk of students and staff during the incorporation of a historically white satellite campus (Rhodes University East London) into a historically black university (University of Fort Hare). The argument, which relies on Essed’s notion of everyday racism, infused with insights from discursive psychology, is that higher education institutions areracialised through the intricate interweaving of macro-level processes and discourses that recur in everyday talk and practices. In their talk, the participants in the study persistently assigned racialised identities to the institutions (Rhodes is white and Fort Hare is black) and invoked a ‘white excellence/black failure’ discourse. ‘White excellence’ folds in on, and is reproduced by, the desirable, modern, urban space and an appeal to Euro-American standards. Institutions and individuals are positioned as being able to overcome ‘black failure’ by moving into white space and through intense personal labour

Cell division in Corynebacterineae

Bacterial cells must coordinate a number of events during the cell cycle. Spatio-temporal regulation of bacterial cytokinesis is indispensable for the production of viable, genetically identical offspring. In many rod-shaped bacteria, precise midcell assembly of the division machinery relies on inhibitory systems such as Min and Noc. In rod-shaped Actinobacteria, for example Corynebacterium glutamicum and Mycobacterium tuberculosis, the divisome assembles in the proximity of the midcell region, however more spatial flexibility is observed compared to Escherichia coli and Bacillus subtilis. Actinobacteria represent a group of bacteria that spatially regulate cytokinesis in the absence of recognizable Min and Noc homologs. The key cell division steps in E. coli and B. subtilis have been subject to intensive study and are well-understood. In comparison, only a minimal set of positive and negative regulators of cytokinesis are known in Actinobacteria. Nonetheless, the timing of cytokinesis and the placement of the division septum is coordinated with growth as well as initiation of chromosome replication and segregation. We summarize here the current knowledge on cytokinesis and division site selection in the Actinobacteria suborder Corynebacterineae

A simulation approach to assessing environmental risk of sound exposure to marine mammals

Intense underwater sounds caused by military sonar, seismic surveys, and pile driving can harm acoustically sensitive marine mammals. Many jurisdictions require such activities to undergo marine mammal impact assessments to guide mitigation. However, the ability to assess impacts in a rigorous, quantitative way is hindered by large knowledge gaps concerning hearing ability, sensitivity, and behavioral responses to noise exposure. We describe a simulation-based framework, called SAFESIMM (Statistical Algorithms For Estimating the Sonar Influence on Marine Megafauna), that can be used to calculate the numbers of agents (animals) likely to be affected by intense underwater sounds. We illustrate the simulation framework using two species that are likely to be affected by marine renewable energy developments in UK waters: gray seal (Halichoerus grypus) and harbor porpoise (Phocoena phocoena). We investigate three sources of uncertainty: How sound energy is perceived by agents with differing hearing abilities; how agents move in response to noise (i.e., the strength and directionality of their evasive movements); and the way in which these responses may interact with longer term constraints on agent movement. The estimate of received sound exposure level (SEL) is influenced most strongly by the weighting function used to account for the specie's presumed hearing ability. Strongly directional movement away from the sound source can cause modest reductions (~5 dB) in SEL over the short term (periods of less than 10 days). Beyond 10 days, the way in which agents respond to noise exposure has little or no effect on SEL, unless their movements are constrained by natural boundaries. Most experimental studies of noise impacts have been short-term. However, data are needed on long-term effects because uncertainty about predicted SELs accumulates over time. Synthesis and applications. Simulation frameworks offer a powerful way to explore, understand, and estimate effects of cumulative sound exposure on marine mammals and to quantify associated levels of uncertainty. However, they can often require subjective decisions that have important consequences for management recommendations, and the basis for these decisions must be clearly described.Publisher PDFPeer reviewe

Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy

Boehm K, Meyer F, Rhomberg A, Kalinowski J, Donovan C, Bramkamp M. Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy. MBIO. 2017;8(3): e00511-17.Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth ratedependent cell cycle models for C. glutamicum. IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum, an emerging cell biological model organism for mycolic acidcontaining bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods

'The difference in determinants of Chlamydia trachomatis and Mycoplasma genitalium in a sample of young Australian women.'

BACKGROUND Differences in the determinants of Chlamydia trachomatis ('chlamydia') and Mycoplasma genitalium (MG) genital infection in women are not well understood. METHODS A cohort study of 16 to 25 year old Australian women recruited from primary health care clinics, aimed to determine chlamydia and MG prevalence and incidence. Vaginal swabs collected at recruitment were used to measure chlamydia and MG prevalence, organism-load and chlamydia-serovar a cross-sectional analysis undertaken on the baseline results is presented here. RESULTS Of 1116 participants, chlamydia prevalence was 4.9% (95% CI: 2.9, 7.0) (n = 55) and MG prevalence was 2.4% (95% CI: 1.5, 3.3) (n = 27). Differences in the determinants were found - chlamydia not MG, was associated with younger age [AOR:0.9 (95% CI: 0.8, 1.0)] and recent antibiotic use [AOR:0.4 (95% CI: 0.2, 1.0)], and MG not chlamydia was associated with symptoms [AOR:2.1 (95% CI: 1.1, 4.0)]. Having two or more partners in last 12 months was more strongly associated with chlamydia [AOR:6.4 (95% CI: 3.6, 11.3)] than MG [AOR:2.2 (95% CI: 1.0, 4.6)] but unprotected sex with three or more partners was less strongly associated with chlamydia [AOR:3.1 (95%CI: 1.0, 9.5)] than MG [AOR:16.6 (95%CI: 2.0, 138.0)]. Median organism load for MG was 100 times lower (5.7 × 104/swab) than chlamydia (5.6 × 10⁶/swab) (p < 0.01) and not associated with age or symptoms for chlamydia or MG. CONCLUSIONS These results demonstrate significant chlamydia and MG prevalence in Australian women, and suggest that the differences in strengths of association between numbers of sexual partners and unprotected sex and chlamydia and MG might be due to differences in the transmission dynamics between these infections.This project was funded by the Commonwealth of Australia, as part of a National Chlamydia Pilot program that is currently running to test the effectiveness of a number of models for chlamydia testing in Australia. This project will assist in developing possible recommendations for a National Chlamydia Program. The analysis of MG was funded by the National Health and Research Council (research grant number 509144)

Prevalent and incident bacterial vaginosis are associated with sexual and contraceptive behaviours in young Australian women

BACKGROUND To determine prevalence and incidence of bacterial vaginosis (BV) and risk factors in young sexually-active Australian women. METHODS 1093 women aged 16-25 years were recruited from primary-care clinics. Participants completed 3-monthly questionnaires and self-collected vaginal smears 6-monthly for 12-months. The primary endpoint was a Nugent Score = 7-10 (BV) and the secondary endpoint was a NS = 4-10 (abnormal flora [AF]). BV and AF prevalence estimates and 95% confidence intervals (95%CI) were derived, and adjusted odds ratios (AOR) calculated to explore epidemiological associations with prevalent BV and AF. Proportional-hazards regression models were used to examine factors associated with incident BV and AF. RESULTS At baseline 129 women had BV [11.8% (95%CI: 9.4-14.2)] and 188 AF (17.2%; 15.1-19.5). Prevalent BV was associated with having a recent female partner [AOR = 2.1; 1.0-4.4] and lack of tertiary-education [AOR = 1.9; 1.2-3.0]; use of an oestrogen-containing contraceptive (OCC) was associated with reduced risk [AOR = 0.6; 0.4-0.9]. Prevalent AF was associated with the same factors, and additionally with >5 male partners (MSP) in 12-months [AOR = 1.8; 1.2-2.5)], and detection of C.trachomatis or M.genitalium [AOR = 2.1; 1.0-4.5]. There were 82 cases of incident BV (9.4%;7.7-11.7/100 person-years) and 129 with incident AF (14.8%; 12.5-17.6/100 person-years). Incident BV and AF were associated with a new MSP [adjusted rate ratio (ARR) = 1.5; 1.1-2.2 and ARR = 1.5; 1.1-2.0], respectively. OCC-use was associated with reduced risk of incident AF [ARR = 0.7; 0.5-1.0]. CONCLUSION This paper presents BV and AF prevalence and incidence estimates from a large prospective cohort of young Australian women predominantly recruited from primary-care clinics. These data support the concept that sexual activity is strongly associated with the development of BV and AF and that use of an OCC is associated with reduced risk.This work was supported by the Commonwealth of Australia, as part of a National Chlamydia Pilot Program and the Australian National Health and Research Council (research grant number 509144). CSB, and JSH were supported by research fellowships issued by the Australian National Health and Medical Research Council (fellowship numbers 456164 and 566576 respectively). MP was supported by a Primary Health Care Research Evaluation and Development MidCareer Fellowship, Department of Health and Aging

Trends in chlamydia and gonorrhea positivity among heterosexual men and men who have sex with men attending a large urban sexual health service in Australia, 2002-2009

<p>Abstract</p> <p>Background</p> <p>To determine whether chlamydia positivity among heterosexual men (MSW) and chlamydia and gonorrhea positivity among men who have sex with men (MSM), are changing.</p> <p>Methods</p> <p>Computerized records for men attending a large sexual health clinic between 2002 and 2009 were analyzed. Chlamydia and gonorrhea positivity were calculated and logistic regression used to assess changes over time.</p> <p>Results</p> <p>17769 MSW and 8328 MSM tested for chlamydia and 7133 MSM tested for gonorrhea. In MSW, 7.37% (95% CI: 6.99-7.77) were chlamydia positive; the odds of chlamydia positivity increased by 4% per year (OR = 1.04; 95% CI: 1.01-1.07; p = 0.02) after main risk factors were adjusted for. In MSM, 3.70% (95% CI: 3.30-4.14) were urethral chlamydia positive and 5.36% (95% CI: 4.82-5.96) were anal chlamydia positive; positivity could not be shown to have changed over time. In MSM, 3.05% (95% CI: 2.63-3.53) tested anal gonorrhea positive and 1.83% (95% CI: 1.53-2.18) tested pharyngeal gonorrhea positive. Univariate analysis found the odds of anal gonorrhea positivity had decreased (OR = 0.93; 95% CI: 0.87-1.00; p = 0.05), but adjusting for main risk factors resulted in no change. Urethral gonorrhea cases in MSM as a percentage of all MSM tested for gonorrhea also fell (p < 0.001).</p> <p>Conclusions</p> <p>These data suggest that chlamydia prevalence in MSW is rising and chlamydia and gonorrhea prevalence among MSM is stable or declining. High STI testing rates among MSM in Australia may explain differences in STI trends between MSM and MSW.</p