Novel protein CcmS is required for stabilization of the assembly of ß-carboxysome in Synechocystis sp. strain PCC 6803.

The carboxysome plays an essential role in the carbon concentration mechanism in cyanobacteria. Although significant progress has been made in the structural analysis of the carboxysome, little is still known about its biosynthesis. We identified slr1911, a gene encoding a protein of unknown function in cyanobacterium Synechocystis sp. Strain PCC 6803 (Syn6803), which we termed ccmS by screening a low CO2 -sensitive mutant. CcmS interacts with CcmK1 and CcmM. The former is a shell protein of the ß-carboxysome and the latter is a crucial component of the ß-carboxysome, which is responsible for aggregating RuBisCO and recruiting shell proteins. The deletion of ccmS lowers the accumulation and assembly of CcmK1, resulting in aberrant carboxysomes, suppressed photosynthetic capacities, and leads to a slow growth phenotype, especially under CO2 -limited conditions. These observations suggest that CcmS stabilizes the assembly of the ß-carboxysome shell and likely connects the carboxysome core with the shell. Our results provide a molecular view of the role played by CcmS in the formation of the ß-carboxysome and its function in Syn6803.

The small RbcS-like domains of the ß-carboxysome structural protein CcmM bind RubisCO at a site distinct from that binding the RbcS subunit.

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO2 escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmMS, joined by unstructured linkers. CcmMS domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first Thermosynechococcus elongatus CcmMS domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmMS, however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmMS domains are able to bind RubisCO in vitro with 1.16 µm affinity. Two or four linked CcmMS domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmMS domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmMS domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmMS bound equally tightly to a RubisCO variant in which the α/ß domain of RbcS was deleted, suggesting that CcmMS binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmMS may bind to an extended electronegative pocket between RbcL dimers.

Salinisphaera aquimarina sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, rod-shaped, motile bacterium with a subpolar flagellum, designated strain CCMM005T, was isolated from offshore seawater at Qingdao, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CCMM005T belonged to the genus Salinisphaera and exhibited highest 16S rRNA gene sequence similarity to Salinisphaera dokdonensis CL-ES53T (96.9 %). It showed lower sequence similarities (94.9-96.4 %) with all other representatives of the genus Salinisphaera. Optimal growth occurred in the presence of 4 % (w/v) NaCl, at 30 °C and at pH 7.0. The polar lipids of strain CCMM005T consisted of phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, one unidentified phosphoglycolipid and one unidentified phospholipid. The predominant isoprenoid quinone was Q-8. The major fatty acids were C19 : 0cyclo ω8c, C18 : 0 and C18 : 1ω7c. The DNA G+C content of strain CCMM005T was 65.3 mol%. On the basis of data from this polyphasic study, strain CCMM005T is considered to represent a novel species of the genus Salinisphaera, for which the name Salinisphaera aquimarina sp. nov. is proposed. The type strain is CCMM005T (=MCCC 1K03246T=KCTC 52640T).

Community perceptions and attitudes on malaria case management and the role of community health workers.

BACKGROUND: Community Case Management of malaria (CCMm) is one of the new approaches adopted by the World Health Organization for malaria endemic countries to reduce the burden of malaria for vulnerable populations. It is based on the evidence that well-trained and supervised community health workers (CHWs) can provide prompt and adequate treatment to fever cases within 24 h to help reduce morbidity and mortality associated with malaria among under-five children. The perception and attitudes of the community members on the CHWs' role is of greater importance for acceptance of their services. The aim of the study was to assess community's perception and attitude towards CCMm and on CHWs who undertake it. METHODS: This study was conducted in five districts in western Kenya where Community Case Management was being undertaken. This was a qualitative cross-sectional study in which in-depth interviews and focus group discussions were conducted with mothers of under-five children and key stakeholders. RESULTS: Overall, there were more positive expressions of perceptions and attitudes of the community members towards the CCMm programme and the role of CHWs. The positive perceptions included among others; recognition and appreciation of services of CHWs, bringing health services to close proximity to the community, avoiding long queues in the health facilities, provision of health education that encourages good health practices, and promotion of positive health-seeking behaviour from within the communities. This programme is not without challenges as some of the negative perceptions expressed by the community members included the fact that some clinicians doubt the capacity of CHWs on dispensing drugs in the community, some CHWs do not keep client's secrets and mistrust of CHWs due to conflicting information by government. CONCLUSIONS: It was evident that the community had more positive perceptions and attitudes towards the role of CHWs in CCMm than negative ones. There should however, be deliberate efforts towards sustaining the positive aspects and addressing the negative concerns raised by the community and the health care practitioners.

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803.

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100µmolphotonsm-2s-1 light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100µmolphotonsm-2s-1 light condition. Under 15µmolphotonsm-2s-1 light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.

Phase Separation of Rubisco by the Folded SSUL Domains of CcmM in Beta-Carboxysome Biogenesis.

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO3- ions into the carboxysome, where they are converted to CO2 by CA. Thus, the carboxysome functions as a CO2-concentrating mechanism (CCM), enhancing the efficiency of Rubisco in CO2 fixation. In ß-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the ß-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions.

Quality of anthropometric data in India's National Family Health Survey: Disentangling interviewer and area effect using a cross-classified multilevel model.

India has adopted a target-based approach to reduce the scourge of child malnourishment. Because the monitoring and evaluation required by this approach relies primarily on large-scale data, a data quality assessment is essential. As field teams are the primary mode of data collection in large-scale surveys, this study attempts to understand their contribution to variations in child anthropometric measures. This research can help disentangle the confounding effects of regions/districts and field teams on the quality of child anthropometric data. The anthropometric z-scores of 2,25,002 children below five years were obtained from the fourth round of India's National Family and Health Survey (NFHS-4), 2015-16. Unadjusted and adjusted standard deviations (SD) of the anthropometric measures were estimated to assess the variations in measurements. In addition, a cross-classified multilevel model (CCMM) approach was adopted to estimate the contribution of geographical regions/districts and teams to variations in anthropometric measures. The unadjusted SDs of the measures of stunting, wasting, and underweight were 1.7, 1.4, and 1.2, respectively. The SD of stunting was above the World Health Organisation threshold (0.8-1.2), as well as the Demographic and Health Survey mark. After adjusting for team-level characteristics, the SDs of all three measures reduced marginally, indicating that team-level workload had a marginal but significant role in explaining the variations in anthropometric z-scores. The CCMM showed that the maximum contribution to variations in anthropometric z-scores came from community-level (Primary Sampling Unit (PSU)) characteristics. Team-level characteristics had a higher contribution to variations in anthropometric z-scores than district-level attributes. Variations in measurement were higher for child height than weight. The present study decomposes the effects of district- and team-level factors and highlights the nuances of introducing teams as a level of analysis in multilevel modelling. Population size, density, and terrain variations between PSUs should be considered when allocating field teams in large-scale surveys.

Binding Options for the Small Subunit-Like Domain of Cyanobacteria to Rubisco.

Two proteins found in cyanobacteria contain a C-terminal domain with homology to the small subunit of rubisco (RbcS). These small subunit-like domains (SSLDs) are important features of CcmM, a protein involved in the biogenesis of carboxysomes found in all ß-cyanobacteria, and a rubisco activase homolog [activase-like protein of cyanobacteria (ALC)] found in over a third of sequenced cyanobacterial genomes. Interaction with rubisco is crucial to the function of CcmM and is believed to be important to ALC as well. In both cases, the SSLD aggregates rubisco, and this nucleation event may be important in regulating rubisco assembly and activity. Recently, two independent studies supported the conclusion that the SSLD of CcmM binds equatorially to L8S8 holoenzymes of rubisco rather than by displacing an RbcS, as its structural homology would suggest. We use sequence analysis and homology modeling to examine whether the SSLD from the ALC could bind the large subunit of rubisco either via an equatorial interaction or in an RbcS site, if available. We suggest that the SSLD from the ALC of Fremyella diplosiphon could bind either in a vacant RbcS site or equatorially. Our homology modeling takes into account N-terminal residues not represented in available cryo-electron microscopy structures that potentially contribute to the interface between the large subunit of rubisco (RbcL) and RbcS. Here, we suggest the perspective that binding site variability as a means of regulation is plausible and that the dynamic interaction between the RbcL, RbcS, and SSLDs may be important for carboxysome assembly and function.