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Problem statement: Rhizobial isolates belonging to genera (Rhizobium sp. and Mesorhizobium sp.) in our laboratory produced only catecholate type of siderophores. Although FhuA and FegA (ferrichrome receptors) homologs were found to be present in the sequenced genomes of few rhizobia (e.g., 1 in R. etli and 2 in Mesorhizobium sp. BNC1), laboratory isolates of the corresponding genera failed to utilize ferrichrome, a siderophore which is present in nanomolar concentrations in the soil. This inability was considered as a negative fitness factor with respect to rhizospheric colonization by these rhizobia. Approach: The 2.4 kb fegA gene (encoding ferrichrome receptor) was amplified along with its native promoter from Bradyrhizobium japonicum 61A152 and cloned in a broad host range plasmid vector pUCPM18. The plasmid construct pFJ was transferred by conjugation into Rhizobium sp. ST1 to give transconjugant ST1pFJ12. The consequence of FegA expression on the transconjugant was tested under lab and soil conditions, using physiological experiments. Results: Ability of the transconjugant ST1pFJ12 to utilize ferrichrome and expression of a 79 kD protein band on the outer membrane of the transconjugant confirmed FegA expression. Transconjugant ST1pFJ12 exhibited increased growth rate as compared to the parent strain ST1, in minimal media containing ferrichrome as the sole iron source, confirming the positive effect of FegA expression. Inoculation of pigeon pea seedlings with transconjugant ST1pFJ12 led to a marked increase in plant growth parameters as compared to plants inoculated with the parent strain ST1, the effect being more pronounced when Ustilago maydis, a ferrichrome producer was co-inoculated in the systems. Nodule occupancy on pigeon pea plant when inoculated with the transconjugant ST1pFJ12 alone was 57% which increased to 66% when co-inoculated with U. maydis as compared with 37 and 30% respectively, seen with parental strain ST1 inoculation. Conclusion: The clear increase in nodule occupancy and higher rhizospheric colonization by the fegA transconjugants, presented in this study together with the previous research reported from our laboratory, led us to conclude that ferrichrome utilization ability played an important role in the rhizospheric colonization of the bioinoculant strains. Testing the ability to utilize hydroxamate siderophores therefore, holds prime importance in selecting an efficient biofertilizer strain. Key words: Ferrichrome, rhizobia, rhizospheric colonization, cross-utilization, nodule occupancy, INTRODUCTION Although iron is the fourth most abundant element on the Earth, it is mainly present in its oxidized state, having a solubility of 10-18M at biological pH[1], which is [...]

Abstract Background Profile Hidden Markov Models (HMM) are statistical representations of protein families derived from patterns of sequence conservation in multiple alignments and have been used in identifying remote homologues with considerable success. These conservation patterns arise from fold specific signals, shared across multiple families, and function specific signals unique to the families. The availability of sequences pre-classified according to their function permits the use of negative training sequences to improve the specificity of the HMM, both by optimizing the threshold cutoff and by modifying emission probabilities to minimize the influence of fold-specific signals. A protocol to generate family specific HMMs is described that first constructs a profile HMM from an alignment of the family's sequences and then uses this model to identify sequences belonging to other classes that score above the default threshold (false positives). Ten-fold cross validation is used to optimise the discrimination threshold score for the model. The advent of fast multiple alignment methods enables the use of the profile alignments to align the true and false positive sequences, and the resulting alignments are used to modify the emission probabilities in the original model. Results The protocol, called HMM-ModE, was validated on a set of sequences belonging to six sub-families of the AGC family of kinases. These sequences have an average sequence similarity of 63% among the group though each sub-group has a different substrate specificity. The optimisation of discrimination threshold, by using negative sequences scored against the model improves specificity in test cases from an average of 21% to 98%. Further discrimination by the HMM after modifying model probabilities using negative training sequences is provided in a few cases, the average specificity rising to 99%. Similar improvements were obtained with a sample of G-Protein coupled receptors sub-classified with respect to their substrate specificity, though the average sequence identity across the sub-families is just 20.6%. The protocol is applied in a high-throughput classification exercise on protein kinases. Conclusion The protocol has the potential to maximise the contributions of discriminating residues to classify proteins based on their molecular function, using pre-classified positive and negative sequence training data. The high specificity of the method, and increasing availability of pre-classified sequence data holds the potential for its application in sequence annotation.

Heme b is an iron-containing co-factor in hemoproteins. Heme b concentrations are low (<1 pmol L -1 ) in iron limited phytoplankton in cultures and in the field. Here, we determined heme b in marine particulate material (>0.7 μm) from the North Atlantic Ocean (GEOVIDE cruise - GEOTRACES section GA01), which spanned several biogeochemical regimes. We examined the relationship between heme b abundance and the microbial community composition, and its utility for mapping iron limited phytoplankton. Heme b concentrations ranged from 0.16 to 5.1 pmol L -1 (median = 2.0 pmol L -1 , n = 62) in the surface mixed layer (SML) along the cruise track, driven mainly by variability in biomass. However, in the Irminger Basin, the lowest heme b levels (SML: median = 0.53 pmol L -1 , n = 12) were observed, whilst the biomass was highest (particulate organic carbon, median = 14.2 μmol L -1 , n = 25; chlorophyll a : median = 2.0 nmol L -1 , n = 23) pointing to regulatory mechanisms of the heme b pool for growth conservation. Dissolved iron (DFe) was not depleted (SML: median = 0.38 nmol L -1 , n = 11) in the Irminger Basin, but large diatoms ( Rhizosolenia sp.) dominated. Hence, heme b depletion and regulation is likely to occur during bloom progression when phytoplankton class-dependent absolute iron requirements exceed the available ambient concentration of DFe. Furthermore, high heme b concentrations found in the Iceland Basin and Labrador Sea (median = 3.4 pmol L -1 , n = 20), despite having similar DFe concentrations to the Irminger Basin, were attributed to an earlier growth phase of the extant phytoplankton populations. Thus, heme b provides a snapshot of the cellular activity in situ and could both be used as indicator of iron limitation and contribute to understanding phytoplankton adaptation mechanisms to changing iron supplies.