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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding 44RKNR47 "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both 44RKNR47 motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.

The vastness of microbial diversity implies that an almost infinite number of individuals needs to be identified to accurately describe such communities. Practical and economical constraints may therefore prevent appropriate study designs. However, for many questions in ecology it is not essential to know the actual diversity but rather the trends among samples thereof. It is, hence, important to know to what depth microbial communities need to be sampled to accurately measure trends in diversity. We used three data sets of freshwater and sediment bacteria, where diversity was explored using 454 pyrosequencing. Each data set contained 615 communities from which 15 00020 000 16S rRNA gene sequences each were obtained. These data sets were subsampled repeatedly to 10 different depths down to 200 sequences per community. Diversity estimates varied with sequencing depth, yet, trends in diversity among samples were less sensitive. We found that 1000 denoised sequences per sample explained to 90% the trends in beta-diversity (Bray-Curtis index) among samples observed for 15 00020 000 sequences. Similarly, 5000 denoised sequences were sufficient to describe trends in a-diversity (Shannon index) with the same accuracy. Further, 5000 denoised sequences captured to more than 80% the trends in Chao1 richness and Pielou's evenness., QC 20120531

Microbial mats are often found in intertidal areas experiencing a large range of salinities. This study investigated the effect of changing salinities on nitrogenase activity and on the composition of the active diazotrophic community ( transcript libraries) of three types of microbial mats situated along a littoral gradient. All three mat types exhibited highest nitrogenase activity at salinities close to ambient seawater or lower. The response to lower or higher salinity was strongest in mats higher up in the littoral zone. Changes in nitrogenase activity as the result of exposure to different salinities were accompanied by changes in the active diazotrophic community. The two stations higher up in the littoral zone showed expression by ( and ) and ( and ). At these stations, a decrease in the relative contribution of to the transcript libraries was observed at increasing salinity coinciding with a decrease in nitrogenase activity. The station at the low water mark showed low cyanobacterial contribution to transcript libraries at all salinities but an increase in deltaproteobacterial transcripts under hypersaline conditions. In conclusion, increased salinities caused decreased nitrogenase activity and were accompanied by a lower proportion of cyanobacterial transcripts.

Cyanobacteria are often the most conspicuous structural part of microbial mats. They are also the only oxygenic phototrophs capable of N2 fixation (diazotrophy). This represents an important advantage for persistence in the often N-depleted marine intertidal microbial mats. In this study the daily pattern of expression of the structural gene for dinitrogenase reductase, nifH and the 16S rRNA gene of 3 benthic filamentous diazotrophic Cyanobacteria (Lyngbya sp., Nodularia sp. and Anabaena sp.) was measured by using quantitative RT-PCR. Gene expression patterns were compared and related to the daily pattern of nitrogenase activity. Microscopic observations revealed that the non-heterocystous Lyngbya sp. was the major cyanobacterial morphotype in all the microbial mats studied. When normalized to copy number, nifH as well as 16S rRNA gene expression by Lyngbya sp. was higher than by the heterocystous Cyanobacteria in all but one mat type. Related to the total amount of extracted RNA, Lyngbya sp. dominated 16S rRNA gene expression as well but was outcompeted by the heterocystous Cyanobacteria with respect to total nifH expression. This was due to the low DNA copy number of Lyngbya sp. nifH. The results revealed different cell-specific expression levels and varying contribution to the overall nifH and 16S rRNA gene expression by the 3 Cyanobacteria over the course of a daily cycle as well as in the different mat types. With respect to the overall nifH expression, the structurally dominant diazotroph was not the most active. Moreover, nifH expression pattern did not follow nitrogenase activity., Cyanobacteria are often the most conspicuous structural part of microbial mats. They are also the only oxygenic phototrophs capable of N2 fixation (diazotrophy). This represents an important advantage for persistence in the often N-depleted marine intertidal microbial mats. In this study the daily pattern of expression of the structural gene for dinitrogenase reductase, nifH and the 16S rRNA gene of 3 benthic filamentous diazotrophic Cyanobacteria (Lyngbya sp., Nodularia sp. and Anabaena sp.) was measured by using quantitative RT-PCR. Gene expression patterns were compared and related to the daily pattern of nitrogenase activity. Microscopic observations revealed that the non-heterocystous Lyngbya sp. was the major cyanobacterial morphotype in all the microbial mats studied. When normalized to copy number, nifH as well as 16S rRNA gene expression by Lyngbya sp. was higher than by the heterocystous Cyanobacteria in all but one mat type. Related to the total amount of extracted RNA, Lyngbya sp. dominated 16S rRNA gene expression as well but was outcompeted by the heterocystous Cyanobacteria with respect to total nifH expression. This was due to the low DNA copy number of Lyngbya sp. nifH. The results revealed different cell-specific expression levels and varying contribution to the overall nifH and 16S rRNA gene expression by the 3 Cyanobacteria over the course of a daily cycle as well as in the different mat types. With respect to the overall nifH expression, the structurally dominant diazotroph was not the most active. Moreover, nifH expression pattern did not follow nitrogenase activity.

The filamentous, non-heterocystous cyanobacterium Microcoleus chthonoplastes is a cosmopolitan organism, known to build microbial mats in a variety of different environments. Although most of these cyanobacterial mats are known for their capacity to fix dinitrogen, M. chthonoplastes has not been assigned as a diazotrophic organism. None of the strains that were correctly identified as M. chthonoplastes has been shown to fix dinitrogen and it has repeatedly been reported that these organisms lacked the cyanobacterial nifH, the structural gene for dinitrogenase reductase. In this study, we show that a complete nif-gene cluster is present in the genome of M. chthonoplastes PCC 7420 and that the three structural nitrogenase genes, nifHDK, are present in a collection of axenic strains of M. chthonoplastes from distant locations. Phylogenetic analysis of nifHDK revealed that they cluster with the Deltaproteobacteria and that they are closely related to Desulfovibrio. The nif operon is flanked by typical cyanobacterial genes, suggesting that it is an integral part of the M. chthonoplastes genome. In this study, we provide evidence that the nif operon of M. chthonoplastes is acquired through horizontal gene transfer. Moreover, the presence of the same nif-cluster in M. chthonoplastes isolates derived from various sites around the world suggests that this horizontal gene transfer event must have occurred early in the evolution of M. chthonoplastes. We have been unable to express nitrogenase in cultures of M. chthonoplastes, but we show that these genes were expressed under natural conditions in the field., The filamentous, non-heterocystous cyanobacterium Microcoleus chthonoplastes is a cosmopolitan organism, known to build microbial mats in a variety of different environments. Although most of these cyanobacterial mats are known for their capacity to fix dinitrogen, M. chthonoplastes has not been assigned as a diazotrophic organism. None of the strains that were correctly identified as M. chthonoplastes has been shown to fix dinitrogen and it has repeatedly been reported that these organisms lacked the cyanobacterial nifH, the structural gene for dinitrogenase reductase. In this study, we show that a complete nif-gene cluster is present in the genome of M. chthonoplastes PCC 7420 and that the three structural nitrogenase genes, nifHDK, are present in a collection of axenic strains of M. chthonoplastes from distant locations. Phylogenetic analysis of nifHDK revealed that they cluster with the Deltaproteobacteria and that they are closely related to Desulfovibrio. The nif operon is flanked by typical cyanobacterial genes, suggesting that it is an integral part of the M. chthonoplastes genome. In this study, we provide evidence that the nif operon of M. chthonoplastes is acquired through horizontal gene transfer. Moreover, the presence of the same nif-cluster in M. chthonoplastes isolates derived from various sites around the world suggests that this horizontal gene transfer event must have occurred early in the evolution of M. chthonoplastes. We have been unable to express nitrogenase in cultures of M. chthonoplastes, but we show that these genes were expressed under natural conditions in the field.

The fixation of nitrogen in cyanobacterial mats situated along the littoral gradient on a Dutch barrier island was investigated by using a high-resolution online, near-real-time acetylene reduction assay. Light-response curves of nitrogenase activity yielded a variety of physiological parameters that changed during a day–night cycle. The fitted parameters were used to calculate nitrogen fixation from the incident natural irradiance over several days in two different mat types. Mats occurring in the higher regions of the littoral were composed of a diverse community of cyanobacteria, consisting of both heterocystous and non-heterocystous filamentous species, whereas closer to the low water mark the mats contained mainly non-heterocystous filamentous cyanobacteria. Although the daily cycles of nitrogenase activity differed considerably between the two types of mats, the daily integrated rates of nitrogen fixation were the same. Moreover, the daily integrated nitrogen fixation seemed to be independent from the daily incident photon flux. The measurements further suggest that different types of diazotrophic cyanobacteria become active at different times of the day and that the composition of the mat community affects maximal and daily patterns of nitrogenase activity. Notwithstanding the apparent light independence of nitrogen fixation, the light-response curves as well as light action spectra unequivocally showed that cyanobacteria were the predominant nitrogen-fixing organisms in these mats. It is concluded that the diversity of nitrogen-fixing cyanobacteria leads to an optimization of this process., The fixation of nitrogen in cyanobacterial mats situated along the littoral gradient on a Dutch barrier island was investigated by using a high-resolution online, near-real-time acetylene reduction assay. Light-response curves of nitrogenase activity yielded a variety of physiological parameters that changed during a day–night cycle. The fitted parameters were used to calculate nitrogen fixation from the incident natural irradiance over several days in two different mat types. Mats occurring in the higher regions of the littoral were composed of a diverse community of cyanobacteria, consisting of both heterocystous and non-heterocystous filamentous species, whereas closer to the low water mark the mats contained mainly non-heterocystous filamentous cyanobacteria. Although the daily cycles of nitrogenase activity differed considerably between the two types of mats, the daily integrated rates of nitrogen fixation were the same. Moreover, the daily integrated nitrogen fixation seemed to be independent from the daily incident photon flux. The measurements further suggest that different types of diazotrophic cyanobacteria become active at different times of the day and that the composition of the mat community affects maximal and daily patterns of nitrogenase activity. Notwithstanding the apparent light independence of nitrogen fixation, the light-response curves as well as light action spectra unequivocally showed that cyanobacteria were the predominant nitrogen-fixing organisms in these mats. It is concluded that the diversity of nitrogen-fixing cyanobacteria leads to an optimization of this process.