Jose V. Lopez

Diversity, structure and convergent evolution of the global sponge microbiome

Sponges (phylum Porifera) are early-diverging metazoa renowned for establishing complex microbial symbioses. Here we present a global Porifera microbiome survey, set out to establish the ecological and evolutionary drivers of these host–microbe interactions. We show that sponges are a reservoir of exceptional microbial diversity and major contributors to the total microbial diversity of the worlds oceans. Little commonality in species composition or structure is evident across the phylum, although symbiont communities are characterized by specialists and generalists rather than opportunists. Core sponge microbiomes are stable and characterized by generalist symbionts exhibiting amensal and/or commensal interactions. Symbionts that are phylogenetically unique to sponges do not disproportionally contribute to the core microbiome, and host phylogeny impacts complexity rather than composition of the symbiont community. Our findings support a model of independent assembly and evolution in symbiont communities across the entire host phylum, with convergent forces resulting in analogous community organization and interactions.

Polyketide Synthase Genes from Marine Dinoflagellates

Rapidly developing techniques for manipulating the pathways of polyketide biosynthesis at the genomic level have created the demand for new pathways with novel biosynthetic capability. Polyketides derived from dinoflagellates are among the most complex and unique structures identified thus far, yet no studies of the biosynthesis of dinoflagellate-derived polyketides at the genomic level have been reported. Nine strains representing 7 different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKSs) by polymerase chain reaction (PCR) and reverse transcriptase PCR. Seven of the 9 strains yielded products that were homologous with known and putative type I PKSs. Unexpectedly, a PKS gene was amplified from cultures of the dinoflagellate Gymnodinium catenatum, a saxitoxin producer, which is not known to produce a polyketide. In each case the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S ribosomal RNA gene. However, amplification from polyadenylated RNA, the lack of PKS expression in light-deprived cultures, residual phylogenetic signals, resistance to methylation-sensitive restriction enzymes, and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes.

Molecular Determination of Species Boundaries in Corals: Genetic Analysis of the Montastraea annularis Complex Using Amplified Fragment Length Polymorphisms and a Microsatellite Marker

Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs, yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata, and rare in M. franksi and M. annularis; in this case homologies were confirmed by Southern hybridizations. A tetranucleotide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique routinely amplifies coral (animal) DNA. Thus analyses based on somatic tissues may be feasible, particularly after diagnostic differences have been established using sperm DNA.

Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp

In this report, real-time quantitative PCR (TaqMan® qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan® qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan® assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Dynamics of marine bacterial community diversity of the coastal waters of the reefs, inlets, and wastewater outfalls of southeast Florida.

Coastal waters adjacent to populated southeast Florida possess different habitats (reefs, oceanic inlets, sewage outfalls) that may affect the composition of their inherent microbiomes. To determine variation according to site, season, and depth, over the course of 1 year, we characterized the bacterioplankton communities within 38 nearshore seawater samples derived from the Florida Area Coastal Environment (FACE) water quality survey. Six distinct coastal locales were profiled – the Port Everglades and Hillsboro Inlets, Hollywood and Broward wastewater outfalls, and associated reef sites using culture‐independent, high‐throughput pyrosequencing of the 16S rRNA V4 region. More than 227,000 sequences helped describe longitudinal taxonomic profiles of marine bacteria and archaea. There were 4447 unique operational taxonomic units (OTUs) identified with a mean OTU count of 5986 OTUs across all sites. Bacterial taxa varied significantly by season and by site using weighted and unweighted Unifrac, but depth was only supported by weighted Unifrac, suggesting a change due to presence/absence of certain OTUs. Abundant microbial taxa across all samples included Synechococcus, Pelagibacteraceae, Bacteroidetes, and various Proteobacteria. Unifrac analysis confirmed significant differences at inlet sites relative to reef and outfalls. Inlet‐based bacterioplankton significantly differed in greater abundances of Rhodobacteraceae and Cryomorphaceae, and depletion of SAR406 sequences. This study also found higher counts of Firmicutes, Chloroflexi, and wastewater associated SBR1093 bacteria at the outfall and reef sites compared to inlet sites. This study profiles local bacterioplankton populations in a much broader context, beyond culturing and quantitative PCR, and expands upon the work completed by the National Oceanic and Atmospheric Administration FACE program.

Diverse bacterial PKS sequences derived from okadaic acid-producing dinoflagellates.

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Population Dynamics of Vibrio spp. Associated with Marine Sponge Microcosms

Vibrio is a diverse genus of marine-associated bacteria with at least 74 species and more expected as additional marine ecospheres are interrogated. This report describes a phylogenetic reconstruction of Vibrio isolates derived from one such unique ecosystem, marine sponges (Phylum Porifera) collected from depths of 150 to 1242 feet. 16S rRNA gene sequencing along with molecular typing of 16S–23S rRNA intergenic spacer regions clustered many sponge-associated Vibrio (spp) with current known species. That is, several benthic Vibrio species commensal with Porifera sponges seemed genetically linked to vibrios associated with coastal or shallow-water communities, signalling a panmictic population structure among seemingly ecologically disparate strains. Conversely, phylogenetic analysis provided evidence for at least two novel Vibrio speciation events within this specific sponge microcosm. Collectively, these findings earmark this still relatively unknown environment as a bastion of taxonomic and phylogenetic variability for the genus and probably other bacterial taxa.

Cooperation and Competition Shape Ecological Resistance during Periodic Spatial Disturbance of Engineered Bacteria

Cooperation is fundamental to the survival of many bacterial species. Previous studies have shown that spatial structure can both promote and suppress cooperation. Most environments where bacteria are found are periodically disturbed, which can affect the spatial structure of the population. Despite the important role that spatial disturbances play in maintaining ecological relationships, it remains unclear as to how periodic spatial disturbances affect bacteria dependent on cooperation for survival. Here, we use bacteria engineered with a strong Allee effect to investigate how the frequency of periodic spatial disturbances affects cooperation. We show that at intermediate frequencies of spatial disturbance, the ability of the bacterial population to cooperate is perturbed. A mathematical model demonstrates that periodic spatial disturbance leads to a tradeoff between accessing an autoinducer and accessing nutrients, which determines the ability of the bacteria to cooperate. Based on this relationship, we alter the ability of the bacteria to access an autoinducer. We show that increased access to an autoinducer can enhance cooperation, but can also reduce ecological resistance, defined as the ability of a population to resist changes due to disturbance. Our results may have implications in maintaining stability of microbial communities and in the treatment of infectious diseases.

The human microbiome: an emerging tool in forensics

Advances in sequencing technology have enabled DNA profiling to become a staple in criminal forensics. Short tandem repeats (STRs) embedded in an individuals’ genetic code enable authorities to take advantage of biological variability to accurately identify and discriminate among people. According to the National DNA Index System (NDIS), CODIS, a DNA database containing more than 12 million profiles, has assisted in more than 340,000 criminal investigations in the USA (CODIS NDIS Statistics, n.d.). However, this still represents a small percentage of total crimes committed. Indeed, many criminal cases still go unsolved despite advances in DNA profiling; for example, in 2015, only 20% of residential burglaries (>1.5 million) were resolved by authorities, according to the 2015 FBI Uniform Crime Reporting statistics (Clearances, n.d.). This can be explained in part by resource allocation, as burglaries are not prioritized for investigation compared to other higher profile crimes (Par e et al., 2007; Coupe, 2016), which can lead to significantly reduced response times, resulting in crime scene evidence contamination or destruction, further impeding investigative efficiency. Therefore, there is a need to improve the lines of evidence that can be acquired to link perpetrators to the crime scene. Improving trace evidence options for criminal investigations is a major focus for forensic research specialists globally. One possible option that has recently emerged encompasses the symbiotic microorganisms that reside in and on our bodies. The NIH-funded ‘Human Microbiome Project’ (HMP) has significantly improved the scientific and public recognition of the vital importance of symbiont ecology to host health and development (Consortium, 2012; Meth e et al., 2012; Grice, 2015). There are approximately as many bacterial cells in our body as human cells (Sender et al., 2016) and the compliment of bacterial taxa, especially at the subspecies level, appears to be unique to each person (Zhu et al., 2015) offering a compelling opportunity to develop a new identifiable marker unique to the individual. The microbiome is even unique in identical twins (Goodrich et al., 2014), theoretically offering an opportunity to increase identity resolution over that possible with human genome evidence. However, the microbiome changes over time in an individual (Oh et al., 2016), so how can it be used to identify a person? While the relative proportions of the bacteria do indeed change, the composition of the community appears to be relatively stable (Caporaso et al., 2011; David et al., 2014), although this stability and continued identifiability are areas of active research. Interestingly, the fluctuations in the structure and composition of the microbiome may contain useful information that could also be used for forensic purposes. Host lifestyle, including diet, occupation, travel, and pharmaceutical use, can influence the composition and structure of microbiome. This suggests that profiling the microbial community in and on our body could also help to reveal details about an individual’s lifestyle (Gonzalez et al., 2016; Kuntz and Gilbert, 2017), which could represent new trace evidence. Profiling the microbiome may be useful in identifying a person or their lifestyle characteristics, but for burglary, the microbiome of the perpetrator would need to be detected at the crime scene, in their absence, while retaining the identifiable characteristics. In support of this, we know that humans shed ~30 million bacterial cells into their vicinity every hour (Qian et al., 2012) and researchers have already demonstrated the forensic potential of the microbiota left behind by people on physical surfaces. For example, the bacterial community found on your finger tips (microbial fingerprint) could be traced on a keyboard, so that which keyboard, and even which keys, a person used could be identified based on the bacterial residue (Fierer et al., 2010). Furthermore, mobile phones carry the personal microbial signatures of the owner (Meadow et al., 2014; Lax et al., 2015). Importantly, these are just preliminary studies, and the results and conclusions cannot be used to justify the application of microbial sequencing to forensic studies. However, the statistical basis for the accurate matching of a person to their microbiota, and evidence that a residual microbial fingerprint could be used to discriminate individuals, does suggest that in the future, it may be possible to use these profiles for forensic investigations. Yet, however intimate the association, because microbial compositions can shift with environmental factors and over time, they cannot be definitively equated

In Vitro Production of Marine-Derived Antitumor Compounds

The sustainable supply of marine-derived bioactive compounds to meet demands for pre-clinical and clinical evaluation is a major consideration in drug development strategies. It is also a concern to environmental resource managers, particularly since conservation of genetic resources may preclude harvesting as a bulk supply strategy. Our research addresses this need by focusing on in vitro production of antitumor compounds through cell culture of the source organisms. Our objectives are: to establish cultures of bioactive marine invertebrates that can be used as models to study in vitro production of antitumor compounds and the factors which control expression of their production; to provide bulk supplies of these compounds through in vitro production; and to produce new structural analogs via manipulation of culture conditions.