Characterization, Selection, and Trans-Species Polymorphism in the MHC Class II of Heermann's Gull (Charadriiformes).

The major histocompatibility complex (MHC) enables vertebrates to cope with pathogens and maintain healthy populations, thus making it a unique set of loci for addressing ecology and evolutionary biology questions. The aim of our study was to examine the variability of Heermann's Gull MHC class II (MHCIIB) and compare these loci with other Charadriiformes. Fifty-nine MHCIIB haplotypes were recovered from sixty-eight Heermann's Gulls by cloning, of them, twelve were identified as putative true alleles, forty-five as unique alleles, and two as pseudogenes. Intra and interspecific relationships indicated at least two loci in Heermann's Gull MHCIIB and trans-species polymorphism among Charadriiformes (coinciding with the documented evidence of two ancient avian MHCIIB lineages, except in the Charadriidae family). Additionally, sites under diversifying selection revealed a better match with peptide-binding sites inferred in birds than those described in humans. Despite the negative anthropogenic activity reported on Isla Rasa, Heermann's Gull showed MHCIIB variability consistent with population expansion, possibly due to a sudden growth following conservation efforts. Duplication must play an essential role in shaping Charadriiformes MHCIIB variability, buffering selective pressures through balancing selection. These findings suggest that MHC copy number and protected islands can contribute to seabird conservation.

Outer Membrane Vesicles of Gram-Negative Bacteria: An Outlook on Biogenesis.

Outer membrane vesicles (OMVs) from Gram-negative bacteria were first described more than 50 years ago. However, the molecular mechanisms involved in biogenesis began to be studied only in the last few decades. Presently, the biogenesis and molecular mechanisms for their release are not completely known. This review covers the most recent information on cellular components involved in OMV biogenesis, such as lipoproteins and outer membrane proteins, lipopolysaccharide, phospholipids, quorum-sensing molecules, and flagella.

Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae.

Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Proteomic Analysis of Membrane Blebs of Brucella abortus 2308 and RB51 and Their Evaluation as an Acellular Vaccine.

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a "vaccine" to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

Emergence of soil bacterial ecotypes along a climate gradient.

The high diversity of soil bacteria is attributed to the spatial complexity of soil systems, where habitat heterogeneity promotes niche partitioning among bacterial taxa. This premise remains challenging to test, however, as it requires quantifying the traits of closely related soil bacteria and relating these traits to bacterial abundances and geographic distributions. Here, we sought to investigate whether the widespread soil taxon Curtobacterium consists of multiple coexisting ecotypes with differential geographic distributions. We isolated Curtobacterium strains from six sites along a climate gradient and assayed four functional traits that may contribute to niche partitioning in leaf litter, the top layer of soil. Our results revealed that cultured isolates separated into fine-scale genetic clusters that reflected distinct suites of phenotypic traits, denoting the existence of multiple ecotypes. We then quantified the distribution of Curtobacterium by analysing metagenomic data collected across the gradient over 18 months. Six abundant ecotypes were observed with differential abundances along the gradient, suggesting fine-scale niche partitioning. However, we could not clearly explain observed geographic distributions of ecotypes by relating their traits to environmental variables. Thus, while we can resolve soil bacterial ecotypes, the traits delineating their distinct niches in the environment remain unclear.

Microdiversity of an Abundant Terrestrial Bacterium Encompasses Extensive Variation in Ecologically Relevant Traits.

Much genetic diversity within a bacterial community is likely obscured by microdiversity within operational taxonomic units (OTUs) defined by 16S rRNA gene sequences. However, it is unclear how variation within this microdiversity influences ecologically relevant traits. Here, we employ a multifaceted approach to investigate microdiversity within the dominant leaf litter bacterium, Curtobacterium, which comprises 7.8% of the bacterial community at a grassland site undergoing global change manipulations. We use cultured bacterial isolates to interpret metagenomic data, collected in situ over 2 years, together with lab-based physiological assays to determine the extent of trait variation within this abundant OTU. The response of Curtobacterium to seasonal variability and the global change manipulations, specifically an increase in relative abundance under decreased water availability, appeared to be conserved across six Curtobacterium lineages identified at this site. Genomic and physiological analyses in the lab revealed that degradation of abundant polymeric carbohydrates within leaf litter, cellulose and xylan, is nearly universal across the genus, which may contribute to its high abundance in grassland leaf litter. However, the degree of carbohydrate utilization and temperature preference for this degradation varied greatly among clades. Overall, we find that traits within Curtobacterium are conserved at different phylogenetic depths. We speculate that similar to bacteria in marine systems, diverse microbes within this taxon may be structured in distinct ecotypes that are key to understanding Curtobacterium abundance and distribution in the environment.IMPORTANCE Despite the plummeting costs of sequencing, characterizing the fine-scale genetic diversity of a microbial community-and interpreting its functional importance-remains a challenge. Indeed, most studies, particularly studies of soil, assess community composition at a broad genetic level by classifying diversity into taxa (OTUs) defined by 16S rRNA sequence similarity. However, these classifications potentially obscure variation in traits that result in fine-scale ecological differentiation among closely related strains. Here, we investigated "microdiversity" in a highly diverse and poorly characterized soil system (leaf litter in a southern Californian grassland). We focused on the most abundant bacterium, Curtobacterium, which by standard methods is grouped into only one OTU. We find that the degree of carbohydrate usage and temperature preference vary within the OTU, whereas its responses to changes in precipitation are relatively uniform. These results suggest that microdiversity may be key to understanding how soil bacterial diversity is linked to ecosystem functioning.

The Genome Sequence of Avibacterium paragallinarum Strain CL Has a Large Repertoire of Insertion Sequence Elements.

The draft genome sequence of Avibacterium paragallinarum strain CL serovar C is reported here. The genome comprises 154 contigs corresponding to 2.4 Mb with 41% G+C content and many insertion sequence (IS) elements, a characteristic not previously reported in A. paragallinarum.

Phenotypic Microdiversity and Phylogenetic Signal Analysis of Traits Related to Social Interaction in Bacillus spp. from Sediment Communities.

Understanding the relationship between phylogeny and predicted traits is important to uncover the dimension of the predictive power of a microbial composition approach. Numerous works have addressed the taxonomic composition of bacteria in communities, but little is known about trait heterogeneity in closely related bacteria that co-occur in communities. We evaluated a sample of 467 isolates from the Churince water system of the Cuatro Cienegas Basin (CCB), enriched for Bacillus spp. The 16S rRNA gene revealed a random distribution of taxonomic groups within this genus among 11 sampling sites. A subsample of 141 Bacillus spp. isolates from sediment, with seven well-represented species was chosen to evaluate the heterogeneity and the phylogenetic signal of phenotypic traits that are known to diverge within small clades, such as substrate utilization, and traits that are conserved deep in the lineage, such as prototrophy, swarming and biofilm formation. We were especially interested in evaluating social traits, such as swarming and biofilm formation, for which cooperation is needed to accomplish a multicellular behavior and for which there is little information from natural communities. The phylogenetic distribution of traits, evaluated by the Purvis and Fritz's D statistics approached a Brownian model of evolution. Analysis of the phylogenetic relatedness of the clusters of members sharing the trait using consenTRAIT algorithm, revealed more clustering and deeper phylogenetic signal for prototrophy, biofilm and swimming compared to the data obtained for substrate utilization. The explanation to the observed Brownian evolution of social traits could be either loss due to complete dispensability or to compensated trait loss due to the availability of public goods. Since many of the evaluated traits can be considered to be collective action traits, such as swarming, motility and biofilm formation, the observed microdiversity within taxonomic groups might be explained by distributed functions in structured communities.

Microevolution Analysis of Bacillus coahuilensis Unveils Differences in Phosphorus Acquisition Strategies and Their Regulation.

Bacterial genomes undergo numerous events of gene losses and gains that generate genome variability among strains of the same species (microevolution). Our aim was to compare the genomes and relevant phenotypes of three Bacillus coahuilensis strains from two oligotrophic hydrological systems in the Cuatro Ciénegas Basin (México), to unveil the environmental challenges that this species cope with, and the microevolutionary differences in these genotypes. Since the strains were isolated from a low P environment, we placed emphasis on the search of different phosphorus acquisition strategies. The three B. coahuilensis strains exhibited similar numbers of coding DNA sequences, of which 82% (2,893) constituted the core genome, and 18% corresponded to accessory genes. Most of the genes in this last group were associated with mobile genetic elements (MGEs) or were annotated as hypothetical proteins. Ten percent of the pangenome consisted of strain-specific genes. Alignment of the three B. coahuilensis genomes indicated a high level of synteny and revealed the presence of several genomic islands. Unexpectedly, one of these islands contained genes that encode the 2-keto-3-deoxymannooctulosonic acid (Kdo) biosynthesis enzymes, a feature associated to cell walls of Gram-negative bacteria. Some microevolutionary changes were clearly associated with MGEs. Our analysis revealed inconsistencies between phenotype and genotype, which we suggest result from the impossibility to map regulatory features to genome analysis. Experimental results revealed variability in the types and numbers of auxotrophies between the strains that could not consistently be explained by in silico metabolic models. Several intraspecific differences in preferences for carbohydrate and phosphorus utilization were observed. Regarding phosphorus recycling, scavenging, and storage, variations were found between the three genomes. The three strains exhibited differences regarding alkaline phosphatase that revealed that in addition to gene gain and loss, regulation adjustment of gene expression also has contributed to the intraspecific diversity of B. coahuilensis.