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Study areaPenang is situated in the northern part of Peninsular Malaysia and lies within the latitudes 5°12’N to 5°30′ N and longitudes 100°09’E to 100°26’E (Fig. 7). Penang with a land area of 295 Km2, has an estimated population of 720,000 and is regarded as the most populated island in Malaysia. Penang shares the same border on the north and east with Kedah State and the south with Perak State. There are two main parts of Penang State: Penang Island and the mainland which is also regarded as Seberang Perai. These two parts of the State are connected by the two bridges. The eastern part of Penang Island is the most urbanized area comprising industries, commercial centres and residential buildings. However, the western part is less developed comprising mainly hilly terrain and forests22. This study is focused on the Island part of Penang. This island is endowed with a yearly equatorial climate (hot and humid). It has a mean annual temperature ranging between 27 and 30 °C while the mean annual relative humidity ranges between 70 and 90%. Also, the mean annual rainfall is about 267–624 cm.Figure 7The map of Penang State showing the Penang Island (created by the authors using ArcMap 10.8 software).Full size imageData acquisitionThe flow chart of the methodology is presented in Fig. 8. Landsat satellite images were used for the assessment of changes in land use covering a period of 2010–2021 (11 years).Figure 8The flow chart of the methodology.Full size imageThese images were gotten from the website of the United State Geological Survey (https://earthexplorer.usgs.gov). The Landsat images include the Landsat 5 TM (thematic mapper) and Landsat 8 OLI / TIRS (operational land imager / thermal infrared sensor). These were downloaded from the Landsat level 1 dataset (Table 6) with additional criteria which reduced the.Table 6 The characteristics of the satellite data used.Full size tableDetermination of LST and NDVI for Landsat 5 and 8Band 6 of Landsat 5 and band 10 of Landsat 8 were used for the determination of the land surface temperature (LST). The LST and normalized difference vegetation index were determined using the following steps:Conversion of top of atmosphere (TOA) radianceUsing the radiance rescaling factor, thermal infra-red digital numbers were converted to TOA spectral radiance using the equation below29: (frac{Red – NIR}{{Red + NIR}}) (frac{Red – NIR}{{Red + NIR}}) For Landsat 8,$$ {text{L}}lambda = left( {{text{ML}} times {text{ Qcal}}} right) + left( {{text{AL}} – {text{Oi}}} right) $$
(1)
For Landsat 5,$$ {text{L}}lambda = left( {{ }frac{{{text{LMax}}lambda – {text{LMin}}lambda }}{{{text{QcalMax}} – {text{QcalMin}}}}} right) times left( {left( {{text{Qcal }} – {text{QcalMin}}} right) + {text{LMin}}lambda } right) $$
(2)
where Lλ is TOA spectral radiance, ML is radiance multiplicative band Number, AL is radiance add band number, Qcal is quantized and calibrated standard product pixel values (DN for band 6 or band 10), Oi is the correction value for the respective bands, LMaxλ is spectral radiance scaled to QcalMax, LMinλ is spectral radiance scaled to QcalMin, QcalMax is maximum quantized calibrated pixel value, and QcalMin is minimum quantized calibrated pixel value.Conversion to TOA brightness temperature (BT)Spectral radiance data were converted to TOA brightness temperature using the thermal constant values in the Metadata file29.Kelvin (K) to Celcius (°C) degrees$$ BT = {raise0.5exhbox{$scriptstyle {K2}$} kern-0.1em/kern-0.15em lower0.25exhbox{$scriptstyle {ln left( {frac{K1}{{{text{L}}lambda { } + { }1}}} right)}$}} – 273.15 $$
(3)
where BT is the Top of atmosphere brightness temperature (°C), Lλ is TOA spectral radiance (W.m−2 .sr−1 .µm−1)), K1 is the K1 constant band number, and K2 is the K2 constant band number. For Landsat 5, K1 is 607.76, and K2 is 1260.56.Normalized difference vegetation index (NDVI)The Normalized Difference Vegetation Index (NDVI) is a standardized vegetation index which reveals the intensity of greenness and surface radiant temperature of the area30,31. The index value of NDVI usually ranges from − 1 to 1. The higher NDVI value indicates that the vegetation of the area is denser and healthier. This shows that the NDVI values of normal healthy vegetation range from 0.1– 0.75, while it is almost zero for rock and soil, and negative value for water bodies24. The NDVI is calculated using the followings:$$ {text{NDVI }} = frac{{left( {{text{NIR }}{-}{text{ RED}}} right){ }}}{{left( {{text{NIR }} + {text{ RED}}} right)}} $$
(4)
In Landsat 4–7$$ {text{NDVI }} = , left( {{text{Band 4 }}{-}{text{ Band 3}}} right) , / , left( {{text{Band 4 }} + {text{ Band 3}}} right) $$In Landsat 8$$ {text{NDVI }} = , left( {{text{Band 5 }}{-}{text{ Band 4}}} right) , / , left( {{text{Band 5 }} + {text{ Band 4}}} right) $$where: RED = DN values from the RED band, and NIR = DN values from the Near Infra-red band.Land Surface Emissivity (LSE)Land Surface Emissivity is the average emissivity of an element on the surface of the earth calculated from NDVI values.$$ {text{PV }} = left{ {frac{{left( {{text{NDVI }} – {text{ NDVImin}}} right)}}{{left( {{text{NDVImax }} – {text{ NDVImin}}} right)}}} right}^{2} $$
(5)
where PV is the Proportion of vegetation, NDVI is the DN value from the NDVI image, NDVImin is the minimum DN value from the NDVI image, and NDVImax is the maximum DN value from the NDVI image.$$ {text{E }} = left( {0.004{ } times {text{PV}}} right) + 0.986 $$
(6)
where E is land surface emissivity, PV is the Proportion of vegetation, 0.986 corresponds to a correction value of the equation.Land Surface Temperature (LST)Land Surface Temperature (LST) is the radiative temperature which is calculated using top of atmosphere brightness temperature, the wavelength of emitted radiance and land surface emissivity.$$ {text{LST}} = {raise0.5exhbox{$scriptstyle {BT}$} kern-0.1em/kern-0.15em lower0.25exhbox{$scriptstyle {left( {1 + left( {{lambda } times { }frac{{{text{BT}}}}{{{text{c}}2}}} right) times {text{ln}}left( {text{E}} right)} right)}$}} $$
(7)
Here c2 is 14388. The value of λ for Landsat 5 (Band 6) is 11.5 µm and Landsat 8 (Band 10) is 10.8 µm.Where BT is the top of atmosphere brightness temperature, λ is the wavelength of emitted radiance, and E is land surface emissivity.c2 = h*c/s (1.4388*10–2 mK = 14388 mK), h is Planck’s constant (6.626*1034 Js), s is Boltzmann constant (1.38*1023 JK), c is velocity of light (2.998*108 m/s).Determination of land use and land cover (LULC) of the study areaThe Landsat images were pre-screened and subjected to clipping and classification32. The boundary shape file of Penang was used to clip out the area of study.Image classificationThe unsupervised method involving a random assignment of sample training points and supervised methods of satellite image classification was employed in this study for determining the LULC types. This mixture of image classification methods has been reported as vital in achieving a high accuracy level33. Bands 5, 4 and 3 were used to classify Landsat 8 while bands 4, 3 and 2 were used for classifying Landsat 5. We used the extraction by mask in the spatial analyst tool of ArcMap 10.2.1 software to extract the study area from the selected bands of the Landsat satellite images. A widely used supervised image classification method was adopted for classifying the Landsat bands in this study32,34. The principle of operation of this method involves the identification of known sample training points which are then used to classify other unknown points with related spectral signatures35. The three monochromatic satellite bands were combined to produce the false colour composite (FCC) using the data management tool36. This involves drawing polygons on the LULC type to select the training points. The LULC types adopted for this study include urbanized areas, agricultural land, rocks, forests, bare surfaces, and water bodies. These were modified LULC types from IPOC Good Practice Guidance37. To achieve this, a minimum of 40 sample points were selected randomly for each category of LULC type36. Having prior knowledge of the study area assisted in the selection of the training points38.The multivariate maximum likelihood classification (MLC) technique was used for transforming the images. Other image transformation techniques have been used by researchers. These include the fuzzy set classifier, neural networks (NN) classifier, extraction and classification of homogenous objects (ECHO) classifier, per-field classifier, sub-pixel classifier, decision trees (DTs), support vector machines (SVMs), minimum distance classifier (MDC) and so-on39. The adoption of any of these techniques is dependent on the knowledge of the area of study, band selection, accessibility of data, the complexity of the landscape, the classification algorithm, and the proficiency of the analyst39. We preferred MLC to other techniques in this study due to its reported high level of accuracy in tropical regions32,34. Another reason for choosing MLC is that it is readily incorporated in many widely used GIS software packages. This MLC algorithm operates based on assigning pixels to the highest probability class and establishing the class ownership of such pixels. It is also regarded as a parametric classifier whose data follows almost a normal distribution39. We ensured the accuracy of this classifier by assigning a large number of training sample points using our prior knowledge of the study area.Description of the LULC categoriesThe urbanized area is the developed part of the study area. This includes houses, roads, railways, and industries. This is also known to be a settlement in other literature40. Agricultural land is the part of the study area dominated by agricultural activities and herbaceous plants and grasses. Agricultural land is generally a product of deforestation36. Rocks are part of the study area comprising solid mineral materials (rocks). Bare land is the bare soil which is either made open by natural or human activities.Forests are parts of the study area dominated by trees. They can be primary or secondary forests depending on the rate of disturbances. According to41, forest land is an area having more than 0.5 ha of flora comprising trees (height is above 5 m) with a canopy greater than 10%. The forests in Penang are generally both primary and secondary42. Water bodies are parts of the study area covered by water seasonally or permanently. These include seas, rivers, lakes, ponds, streams, or reservoirs40.Determination of change in the LULCThe rate and extent of change in the LULC of Penang within the periods under consideration were determined following the formula below43:$$ {text{Changed area }}left( {{text{C}}_{{text{a}}} } right) , = {text{ T}}_{{text{a}}} left( {text{year 2}} right) , {-}{text{ T}}_{{text{a}}} left( {text{year 1}} right) $$
(8)
$$ {text{Changed extent }}left( {{text{C}}_{{text{e}}} } right) , = {text{ C}}_{{text{a}}} /{text{ T}}_{{text{a}}} left( {text{year 1}} right) $$
(9)
$$ {text{Percentage of change }} = {text{ C}}_{{text{e}}} {text{x 1}}00 $$
(10)
where Ta means the total area.Determination of relationship between LST and NDVIThe values of LST and NDVI at 20 random points of each LULC class were used. The relationship between the LST and NDVI across all the LULC classes in each year was determined using the bivariate linear regression analysis. This was done in Paleontological Statistical (PAST) package 3.0.Classification accuracy assessmentThe classification accuracy was assessed by taking ground truth coordinate data of the LULC of the study area using a geographical positioning system (GPS) device (Garmin Etrex 10). These data were compared with the LULC classified in this study32. Consequently, an error matrix was generated. This normally uses ground truth data to explain the accuracy of the classified LULC. The error matrix comprises the user’s accuracy, the producer’s accuracy, overall accuracy and the Kappa index32.The producer’s accuracy (omission error) represents the probability of the correctly classified reference pixel and it is determined using this formula below:$${text{Producer’s accuracy }}left( % right) , = { 1}00% , – {text{ error of omission}} $$
(11)
Also, the user’s accuracy (commission error) represents the probability that the classified pixel matches the one on the ground36 and it is determined using the formula below:$$ {text{User’s accuracy }}left( % right) , = { 1}00% , – {text{ error of commission}} $$
(12)
The statistical accuracy of the matrix was determined using the Kappa coefficient44. This Kappa coefficient ranges from − 1 to + 145. Therefore, the overall accuracy of the classification was determined by dividing the total number of correctly classified pixels by the total number of sampled ground truth data40. More

RESEARCH BRIEFINGS
07 December 2022

Ancient environmental DNA from northern Greenland opens a new chapter in genetic research, demonstrating that it is possible to track the ecology and evolution of biological communities two million years ago. The record shows an open boreal-forest ecosystem inhabited by large animals such as mastodons and reindeer. More

Restoring the connection between people and the rest of nature hinges on whole-system science, actions and negotiations.
Those who think about and practise sustainability are constantly looking for holistic interpretations of the world and are trying to understand systemic relations, networks and connections. Biodiversity has all of these things. It shows how every species needs other species to exist and thrive. It shows that all living organisms are part of a sophisticated and fascinating system made up of myriads of links. And humans are undoubtedly a part of it.
Credit: Pulsar Imagens / Alamy Stock PhotoIn the realm of sustainability, experts also ponder about time: how can life exist and thrive over time? Indeed, the above mentioned fascinating system evolves over time. And, over time, it has to adapt to unexpected change. It does that well when it is healthy, and less well when it is ill and constantly disturbed.For a long time, man-made impacts kept accumulating almost completely unchecked by societies, until the consequences for human well-being became untenable. Nowadays, environmental crises make the headlines regularly. They are nothing but the result of a broken connection between people and the rest of nature.Climate change is one major outcome of the broken human–rest of nature connection and has wide ramifications for both people and the planet. We now face imminent disaster, unequally across the world, yet addressing climate change remains an incredibly thorny task. Country representatives from most nations around the world meet regularly at the Conference of the Parties (COP) to the United Nations Framework Convention on Climate Change (UNFCC) — most recently at COP27, which was held in Egypt — to continue the debate on what actions are needed to move the climate agenda forward, all while disasters continue to hit the most vulnerable populations. The world has seen 27 COP meetings to the UNFCC so far; one wonders how many more meetings will be needed to see real change happen.Interestingly, country representatives also meet regularly to discuss biodiversity protection; biodiversity decline — the other major consequence of the broken human–rest of nature connection — is just as worrying, with severe and ramified implications that are still largely underappreciated by decision-makers. These gatherings are the COP meetings to the Convention on Biological Diversity (CBD). Last year, we wrote about the then forthcoming COP15 to the CBD (Nat. Sustain. 4, 189; 2021), the meeting in which the new conservation targets to be met by 2030 were to be agreed. We highlighted the extent to which experts worried that those new targets might not go far enough. The meeting was postponed more than once due to the COVID-19 pandemic, and it is finally happening on 7 December 2022, in Montreal, Canada. The world has already seen 15 COP meetings to the CBD, how many more meetings will be needed for the biodiversity crisis to be averted?But let’s go back to thinking about sustainability. Experts look for holistic visions of the world. Here is an interesting example of what holism means. Biodiversity decline and climate change are both the result of the broken connection between people and the rest of nature, they ultimately have the same, deep roots. They are mutually reinforcing phenomena: unhealthy biodiversity contributes to climate change, and climate change makes biodiversity ill. All this is bad news for human and planetary well-being. The climate–biodiversity conundrum, at least to some degree, has been recognized at a higher level — during COP27, leaders dedicated one day to biodiversity.Yet, given that these issues are highly interconnected and have the same origin, why is the world insisting on discussing them as separate agendas? Why are we still holding two separate COPs? How are these meetings going to promote any fruitful synergy? How will they lead people to reconnect with the rest of nature? Country representatives should be breaking silos, embracing holism and bringing these intertwined issues, and their multiple ramifications, to the same negotiating table.Nature Sustainability welcomes the long-awaited COP15 to the CBD and hopes that countries will agree on feasible yet ambitious 2030 targets to protect and enhance biodiversity. But most of all, we hope that all of the experts and leaders involved in addressing the environmental crises embrace holism to promote meaningful actions across the world aimed at restoring people’s connection with the rest of nature. We are eager to see progress to this end. In the meantime, the collection we started in March 2021 with Nature Ecology & Evolution has been updated to renew our support to the biodiversity community. More

Predictors of reservoir statusOur analyses include all known rodent reservoirs for zoonotic pathogens (282 species). These reservoirs harbour a total of 95 known zoonotic pathogens (34 viruses, 26 bacteria, 17 helminths, 12 protozoa and six fungi) employing all known modes of transmission (43 vector-borne, 32 close-contact, 28 non-close contact, and 13 using multiple transmission modes) (Supplementary Data 2). Compared to presumed non-reservoirs (species currently not known to harbour any zoonotic pathogens), we observed that reservoir rodents are strikingly synanthropic (Figs. 2, 3a, Table 1). Despite potential geographic biases, and the general possibility that synanthropic species are better studied compared to non-synanthropic species (see Sampling bias and Supplementary Figs. 1, 2), synanthropy emerged as a defining characteristic of nearly all (95%) currently known rodent reservoirs. Of the 155 synanthropic species, only six are considered as truly synanthropic, i.e., predominately, if not exclusively, occurring in or near human dwellings, while the remaining species only occasionally show synanthropic behaviour (Supplementary Data 1).Fig. 2: Predictors of reservoir status.Final structural equation model linking reservoir status of rodent species (n = 269) with their synanthropy and hunting status, population fluctuations (s-index, log-transformed), and adult body mass, controlling for their occurrence in a range of habitats and the number of studies available per species. One-sided (directional) arrows represent a causal influence originating from the variable at the base of the arrow, with the width of the arrow and associated value representing the standardised strength of the relationship. The small double-sided arrows and numbers next to each response (endogenous) variable represent the error variance.Full size imageFig. 3: Characteristics of reservoir and synanthropic rodents.a Reservoir rodents are predominately synanthropic (n = 436 with n (non-reservoir) = 154, n (reservoir) = 282). b Synanthropic rodents display high population fluctuations (high s-index) (n = 269) and c, occur in multiple artificial habitats (n = 269) (Tables 1–3). In a, estimated probability and 95% confidence intervals are shown and in b–c, estimated probability is shown and shaded areas show 95 % confidence intervals.Full size imageTable 1 Summary of best-fit generalized linear mixed effects model for reservoir status (n = 436)Full size tableCompared to non-reservoirs, we also found that rodent reservoirs are disproportionately exploited by humans (hunted for meat and fur). Seventy-two of the regularly hunted rodent species (n = 83) are reservoirs (87%), and hunted rodent species harbour on average five times the number of zoonotic pathogens than non-hunted species (Table 2).Table 2 Summary of rodent characteristics divided by rodent group with respect to hunting, reservoir status, and synanthropic behaviourFull size tableWe explored causal pathways using a structural equation model (SEM) linking synanthropy, reservoir status, and their hypothesized predictors. The final model, which we established a priori, had 17 free parameters and 21 degrees of freedom (n = 269). The model fit, based on the SRMR (standardized root mean squared residual) and the RMSEA (root mean squared error of approximation) indicated a good fit (see Methods). From the initially formulated full model, the pathways linking reservoir status to population fluctuations (s-index, Methods), occurrence in grasslands, number of artificial habitats a species occurs in, and number of studies found per species were not significant and thus removed from the final model (Supplementary Fig. 3). Similarly, pathways linking synanthropy and occurrence in grasslands were not significant and also removed. All reported coefficients for pathways are standardized to facilitate comparisons among the different relationships. The relationships and coefficients below all refer to those in the final model.The focal variable in the model was reservoir status, which was strongly and positively associated with synanthropy and had the highest estimated pathway coefficient (standardised estimate = 0.58, 95% CI 0.49–0.66, Fig. 2). Controlling for synanthropy, species were more likely to be a reservoir with increasing adult weight (0.13, 0.04–0.22). Species that occur in savanna were less likely to be reservoirs (−0.13, −0.22 to −0.04), while hunted species were more likely to be reservoirs (Fig. 2, 0.20, 0.11–0.30).Synanthropy was influenced by four habitat variables: a species was more likely to be synanthropic if it occurs in a higher number of artificial habitats (0.17, 0.04–0.31), and occurs in urban areas (0.14, 0.01–0.27), deserts (0.12, 0.01–0.23), or forests (0.13, 0.02–0.24). Notably, species with higher s-index, and thus larger population fluctuations, were more likely to be synanthropic (0.12, 0.01–0.22), and the s-index itself decreased as adult weight increased (−0.16, −0.27 to −0.04). Finally, hunted species were characterized by higher adult bodyweight (0.35, 0.25–0.44) (Fig. 2).The number of studies per species was positively associated with both a species’ synanthropic behaviour (0.29, 0.19–0.39) and its reservoir status (0.09, 0.00– 0.19), albeit with weaker evidence for the latter effect (p = 0.054) (Fig. 2),The confirmatory generalized linear mixed effects models (GLMMs) (Tables 1, 3), which control for correlation among species within the same family, showed that our SEM results were robust. Indeed, synanthropy was a significant predictor of reservoir status. These models underscore synanthropy as the most important predictor of reservoir status in our analysis (Table 1, Figs. 2–3).Table 3 Summary of best-fit generalized linear mixed effects model for synanthropic status (n = 269)Full size tablePopulation fluctuations affect transmission riskOur newly compiled data on the magnitude of population fluctuations enabled comparative investigations beyond theoretically straightforward predictions that transmission risk increases with reservoir abundance for density-dependent systems. We show that while strong population fluctuations (measured as the s-index) are found frequently in both reservoir and non-reservoir rodents (Table 2), synanthropic rodents exhibit much larger population fluctuations compared to non-synanthropic rodents (Table 2, Figs. 2–3). This pattern was apparent despite broad confidence intervals in the relationship between the s-index and the probability of being synanthropic (Fig. 3b, Tables 2, 3). Taken together, our results suggest that larger population fluctuations in reservoir species increase zoonotic transmission risk via synanthropic behaviours of rodents, thereby increasing the likelihood of zoonotic spillover infection to humans.Habitat generalism and habitat transformation increase transmission riskWe also find that reservoir species thrive in human-created (artificial) habitats (Fig. 3a, c, Tables 2–3), which reflects a general flexibility in their use of diverse habitat types compared to non-reservoir species (Fig. 4a, Table 2). In addition, the number of zoonotic pathogens harboured by a rodent species increased with habitat breadth (r436 = 0.34, p  More

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In this episode:00:45 World’s oldest DNA shows that mastodons roamed ancient GreenlandDNA recovered from ancient permafrost has been used to reconstruct what an ecosystem might have looked like two million years ago. Their work suggests that Northern Greenland was much warmer than the frozen desert it is today, with a rich ecosystem of plants and animals.Research Article: Kjær et al.Nature Video: The world’s oldest DNA: Extinct beasts of ancient Greenland08:21 Research HighlightsWhy low levels of ‘good’ cholesterol don’t predict heart disease risk in Black people, and how firework displays affect the flights of geese.Research Highlight: ‘Good’ cholesterol readings can lead to bad results for Black peopleResearch Highlight: New Year’s fireworks chase wild geese high into the sky10:31 Modelling the potential emissions of plasticsWhile the global demand for plastics is growing, the manufacturing and disposal of these ubiquitous materials is responsible for significant CO2 emissions each year. This week, a team have modelled how CO2 emissions could vary in the context of different strategies for mitigating climate change. They reveal how under specific conditions the industry could potentially become a carbon sink.Research Article: Stegmann et al.News and Views: Plastics can be a carbon sink but only under stringent conditionsSubscribe to Nature Briefing, an unmissable daily round-up of science news, opinion and analysis free in your inbox every weekday.Never miss an episode. Subscribe to the Nature Podcast on Apple Podcasts, Google Podcasts, Spotify or your favourite podcast app. An RSS feed for Nature Podcast is available too. More

Bacterial strains and cultivationAll marine bacteria used in this study were cultivated using the ½ YTSS (yeast-tryptone-sea salt) medium (DSMZ 974), containing yeast extract 2 g/L, tryptone 1.25 g/L and Sigma sea salts 20 g/L or the defined marine ammonium mineral salt (MAMS) medium (DSMZ 1313) where HEPES (10 mM, pH 8.0) replaced the phosphate buffer [16]. All cultures were grown at 30 °C aerobically in a shaker (150 rpm).For growth competition assays between the WT and the olsA mutant, cultures of bacteria were grown in 10 mL ½ YTSS medium for the WT strain, or with the addition of 10 µg/mL gentamicin for the olsA mutant since a gentamicin cassette was inserted to construct the mutant [4]. Cells were harvested at mid-late exponential phase and diluted to an optical density measured at 540 nm (OD540) of 1.0. These cells were then both inoculated at 1% (v/v) into 250 mL flasks containing 50 mL growth media (either ½ YTSS or MAMS + 0.5 mM Pi) in triplicate and grown at 30 °C with shaking at 140 rpm. At time point 0 h, 100 µL samples were removed in triplicate from each flask. These samples were then ten-fold serially diluted in the same growth media to a dilution of 10−9. From each serial dilution tube, 10 µL droplets were pipetted in triplicate onto agar plates containing either ½ YTSS agar (to count both the WT and the olsA mutant) or ½ YTSS agar + 10 µg/mL gentamicin (to count just the olsA mutant). Once the droplets were dry, plates were incubated at 30 °C for 3-4 days. Colony forming units (CFU) were determined by counting the number of colonies in the dilution number where single colonies were clearly visible. For the cultures grown in ½ YTSS medium, samples were removed and enumerated using the same method at time points 24 h and 96 h. For the cultures grown in MAMS media + 0.5 mM Pi, samples were removed and enumerated at time points 0 h, 48 h and 96 h.Membrane separation by sucrose density gradient ultracentrifugationThe WT strain and the olsA mutant were grown in ½ YTSS medium to OD540 ~0.8. One litre of culture was then collected by centrifugation at 12,300 × g at 4 °C for 10 minutes, using a JLA 10.5 rotor. Cells were washed and resuspended in 50 mL HEPES buffer (pH 8.0, 10 mM). Cells were then pelleted by centrifugation at 4,500 × g at 4 °C for 10 min, before resuspending the pellet in 3 mL HEPES buffer (pH 8.0, 10 mM), containing 1.6X cOmplete Protease Inhibitor cocktail (Roche), 3X DNAse I buffer (NEB) and 6 units/mL DNase I (NEB). Cells were then lysed using a French Press at 1000 PSI. Cell debris was removed by centrifugation at 4,500 × g at 4 °C for 10 min and the supernatant was transferred to a new Oakridge centrifuge tube for pelleting total membranes by centrifugation at 75,600 × g at 10 °C for 45 min in a JA25.5 rotor. Pelleted membranes were then washed and resuspended in 20% (w/v) sucrose in HEPES buffer (10 mM, pH 8.0). Resuspended membrane samples were then layered on top of a stepwise gradient containing 3.3 mL 73% (w/v) sucrose at the bottom and 6.7 mL 53% (w/v) sucrose in between. Inner (IM) and outer (OM) membranes were separated by centrifugation at 140,000 × g at 4 °C, for 16 hours in a SW40-Ti rotor. The IM resided in the interface between the 53% (w/v) and 20% (w/v) sucrose layers and the OM in the interface between the 53% (w/v) and 73% (w/v) sucrose layers. Both IM and OM samples were removed from the sucrose density interface, diluted with 30 mL HEPES buffer (10 mM, pH 8.0), and pelleted by centrifugation at 75,600 × g for 45 min. IM and OM were then resuspended in 1 mL of the same HEPES buffer before lipid and protein extractions.Proteomics sample preparation, in-gel digestion and nanoLC-MS analysisIM and OM samples were carefully dissolved in 100 μL 1X LDS loading buffer (Invitrogen) before loading on a precast Tris-Bis NuPAGE gel (Invitrogen) using 1X MOPS running solution (Invitrogen). SDS-polyacrylamide gel electrophoresis was run for approximately 5 min to purify polypeptides in the polyacrylamide gel by removing contaminants. Polyacrylamide gel bands containing the membrane proteome were excised and digested by trypsin (Roche) proteolysis. The resulting tryptic peptides were extracted using formic acid-acetonitrile (5%:25%, v/v) before resuspension in acetonitrile-trifluoroacetate (2.5%:0.05%, v/v). Tryptic peptides were separated by nano-liquid chromatography (nanoLC) using an Ultimate 3000 LC system with an Acclaim PepMap RSLC C18 reverse phase column (ThermoFisher) at the Proteomics Research Technology Platform (PRTP) at the University of Warwick. MS/MS spectra were collected using an Orbitrap Fusion mass spectrometer (ThermoFisher) in electrospray ionization (ESI) mode. Survey scans of peptides from m/z 350 to 1500 were collected for each sample in a 1.5-hr LC-MS run. This resulted in 12 mass spectra (3 biological replicates of IM and OM of WT and the olsA mutant) with a total of ~ 7.5 G of MS/MS data.MS/MS data search and statistical analysesCompiled MS/MS raw files were searched against the genome of Ruegeria pomeroyi DSS-3 using the MaxQuant software package [17, 18]. Default settings were used and samples were matched between runs. The software package Perseus (v1.6.5.0) was used to determine differentially expressed proteins with a false discovery rate (FDR) of 0.01 [19]. The LFQ (label-free quantitation) intensity of each protein was normalized by dividing the total peptide intensity of each sample by the length of each protein. Peptides were retained for further analyses only if they were consistently found in all three biological replicates in at least one set of the four samples (IM_WT, IM_olsA, OM_WT, OM_olsA). Missing values were imputed using the default parameters (width, 0.3; down-shift 1.8) and statistical analyses were performed using a two-sample Student’s t-test. Principle component analysis (PCA) plots and volcano plots were generated using default settings in the Perseus package.To analyse the pathways of differentially expressed proteins between the wild-type and the mutant, the sequences of those proteins that were significantly overrepresented (FDR  More