Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)]Keywords: Photobacterium · taxonomy · symbiosis · pathogenesis · virulence factor

Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M . W . B eijerinck

Luminous bacteria isolated by M artinus W . B eijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names P hotobacterium phosphoreum and ‘ P hotobacterium splendidum ’. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The B eijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi‐locus sequence analysis, based on the 16S rRNA , gapA , gyrB , pyrH , recA , luxA , and luxB genes, revealed that the B eijerinck strains are distant from the type strains of P . phosphoreum , ATCC 11040 T , and V ibrio splendidus , ATCC 33125 T , and instead form an evolutionarily distinct clade of V ibrio . Newly isolated strains from coastal seawater in N orway, F rance, U ruguay, M exico, and J apan grouped with the B eijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram‐negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88047/1/fem1177.pd

Use of LuxA sequences for investigation on Luciferases kinetics and characterization of luminous bacteria

Known luminous bacteria belong to the genera Vibrio, Photobacterium, Shewanella, and Photorhabdus. The enzyme luciferase catalyzing the luminous reaction is composed by the α and β polypeptides and subunit α is responsible for substrate binding and catalytic activities. Luciferases are classified into slow of Vibrio harveyi and fast of Photobacterium sps. on the basis of enzyme kinetics. Shewanella woodyi has intermediate kinetics. This research has tested the hypothesis of existence of three kinetic classes by sequencing luxA gene (coding for c subunit) of new strains and comparing these clusters to phenotypic analysis and sequencing of 16S rRNA. Phenotypic analysis has shown strains distinct from the known. LuxA amino acids and nucleotides and 16S rRNA sequences have shown 5 major lineages corresponding to known species. A dlade distinct from the known species was present. Geographic location and fish habitat didn\u27t affect the distribution of strains

Bioluminescent Fish, Bacteria, and Experiential Learning

Bioluminescence is the process by which living organisms emit light, and it has a large present in marine organisms. Specifically, marine fishes utilize bioluminescence (self-produced or achieved by creating a symbiosis with bioluminescent bacteria) for camouflage, communication, etc. The three central questions of my thesis are: What is the morphology of the lestidiid light organ (a group of luminescent deep sea fish demonstrating counterillumination), and how does this information inform the evolutionary phylogeny of this clade? Are quorum quenching genes present in Photobacterium leiognathi (luminescent bacterium symbionts of ponyfishes, glowbellies, etc.), and how does sequence information inform host specificity and the evolutionary phylogeny of P. leiognathi strains? Lastly, what is experiential learning, how does it tie into scientific research (how did research become such an important part of the university), and how does this style of learning apply to the two experiments I conducted? I conducted extensive background research on these three topics to inform my studies, then conducted the experiments using gross dissection and histology (lestidiid light organ research) as well as DNA extraction, amplification, and sequencing techniques (lestidiid and P. leiognathi research). Lestidiid light organs are shown to derive from hepatopancreatic tissue that was coopted to act as luminescent tissue. P. leiognathi lack the quorum quenching gene of interest, but the phylogeny I constructed with my data and published data adds credibility to the high degree of host specificity between bacterial strains and host fish species. Lastly, I discuss how the search for empirical knowledge propelled the development of scientific research in the university, for questions are an integral and everlasting part of the scientific process

Lateral Replacement of the Lux Operon in a Vibrio Isolated from the Intestine of a Coral Reef Fish

In a screening of bioluminescent bacteria isolated from the intestines of coral reef fish, two strains (designated D6 and M1) were identified that have a luxA gene sequence significantly different from those of other Vibrio species. Phylogenetic analysis of several housekeeping genes, as well as toxR, shows that D6 and M1 branch within a bioluminescent clade (designated the “D1 group,” isolated at the same time and place as D6 and M1) that is a close sister group to Vibrio harveyi. However, whereas the luxA genes of the D1 group are \u3e98% identical to V. harveyi luxA, the luxA genes of D6 and M1 have a surprisingly low identity (86%) to the D1 group and to V. harveyi. Strain D6 and strain D1 (a representative of the D1 group) were chosen for further investigation. The lux operons (luxCDABEGH) and flanking regions of both strains were cloned into E. coli and sequenced by primer walking. Although distinguishable from Vibrio harveyi, and possibly representing a new species, strain D1 is clearly a close relative, and has the same genes flanking the lux operon as V. harveyi. However, in addition to a highly divergent lux operon, the flanking regions of D6 are completely different from those of D1 and V. harveyi. Based on differences in luxCDABEGH sequence and chromosomal context, we conclude that the lux operon of D6 was acquired by lateral gene transfer. PCR and Southern hybridizations show that D6 contains a single lux operon, so we conclude that this operon represents not simply a lateral transfer, but a lateral replacement of the original operon. We also show, in an E. coli expression system, that the lux operons of both D1 and D6 are up-regulated by the V. harveyi LuxR protein, indicating evolutionary conservation of lux gene regulation, despite the high degree of sequence dissimilarity between the two. These results show that we have not exhausted the diversity of bioluminescence genes in bacteria

Phylogenetic analysis of host–symbiont specificity and codivergence in bioluminescent symbioses

Several groups of marine fishes and squids form mutualistic bioluminescent symbioses with luminous bacteria. The dependence of the animal on its symbiont for light production, the animal's specialized anatomical adaptations for harboring bacteria and controlling light emission, and the host family bacterial species specificity characteristic of these associations suggest that bioluminescent symbioses are tightly coupled associations that might involve coevolutionary interactions. Consistent with this possibility, evidence of parallel cladogenesis has been reported for squid–bacterial associations. However, genetic adaptations in the bacteria necessary for and specific to symbiosis have not been identified, and unlike obligate endosymbiotic associations in which the bacteria are transferred vertically, bacterially bioluminescent hosts acquire their light-organ symbionts from the environment with each new host generation. These contrasting observations led us to test the hypotheses of species specificity and codivergence in bioluminescent symbioses, using an extensive sampling of naturally formed associations. Thirty-five species of fish in seven teleost families (Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae, Monocentridae, Acropomatidae, Leiognathidae) and their light-organ bacteria were examined. Phylogenetic analysis of a taxonomically broad sampling of associations was based on mitochondrial 16S rRNA and cytochrome oxidase I gene sequences for the fish and on recA , gyrB and luxA sequences for bacteria isolated from the light organs of these specimens. In a fine-scale test focused on Leiognathidae, phylogenetic analysis was based also on histone H3 subunit and 28S rRNA gene sequences for the fish and on gyrB , luxA , luxB , luxF and luxE sequences for the bacteria. Deep divergences were revealed among the fishes, and clear resolution was obtained between clades of the bacteria. In several associations, bacterial species identities contradicted strict host family bacterial species specificity. Furthermore, the fish and bacterial phylogenies exhibited no meaningful topological congruence; evolutionary divergence of host fishes was not matched by a similar pattern of diversification in the symbiotic bacteria. Re-analysis of data reported for squids and their luminous bacteria also revealed no convincing evidence of codivergence. These results refute the hypothesis of strict host family bacterial species specificity and the hypothesis of codivergence in bioluminescent symbioses. © The Willi Hennig Society 2007.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73754/1/j.1096-0031.2007.00157.x.pd

The Role of Bacterial Symbionts and Bioluminescence in the Pyrosome, Pyrosoma atlanticum

The pelagic tunicate, Pyrosoma atlanticum, is known for its brilliant bioluminescence, but the mechanism causing this bioluminescence has not been fully characterized. This study identifies the bacterial bioluminescent symbionts of P. atlanticum collected in the northern Gulf of Mexico using various methods such as electron microscopy, light microscopy, and molecular genetics. The bacteria are localized within a specific pyrosome light organ. Bioluminescent symbiotic bacteria of Vibrionaceae composed \u3e50% of taxa in tunicate samples (n=13), which was shown by utilizing current molecular genetics methodologies. While searching for bacterial lux genes in 2 tunicate samples, we also serendipitously generated a draft tunicate mitochondrial genome which was used for P. atlanticum pyrosome identification. Furthermore, a total of 396K MiSeq16S rRNA reads provided pyrosome microbiome profiles to determine bacterial symbiont taxonomy. After comparing with the Silva rRNA database, a 99% sequence identity matched a Photobacterium sp. R33-like bacterium (referred to as Photobacterium-Pa1) as the most abundant bacteria within P. atlanticum samples. Specifically-designed 16S rRNA V4 probes for fluorescence in situ hybridization (FISH) verified the Photobacterium-Pa1 location around the periphery of each pyrosome luminous organ. Scanning and transmission electron microscopy (SEM, TEM respectively) confirmed a rod-like bacterial presence which also appears intracellular in the light organs. This intracellular bacterial localization may represent a bacteriocyte formation reminiscent of other invertebrates

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium

Identification and Signature Sequences of Bacterial Δ4,5Hexuronate-2-O-Sulfatases

Glycosaminoglycan (GAG) sulfatases, which catalyze the hydrolysis of sulfate esters from GAGs, belong to a large and conserved sulfatase family. Bacterial GAG sulfatases are essential in the process of sulfur cycling and are useful for the structural analysis of GAGs. Only a few GAG-specific sulfatases have been studied in detail and reported to date. Herein, the GAG-degrading Photobacterium sp. FC615 was isolated from marine sediment, and a novel Δ4,5hexuronate-2-O-sulfatase (PB2SF) was identified from this bacterium. PB2SF specifically removed 2-O-sulfate from the unsaturated hexuronate residue located at the non-reducing end of GAG oligosaccharides produced by GAG lyases. A structural model of PB2SF was constructed through a homology-modeling method. Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis. N113A, K141A, K141H, H143A, H143K, H205A, and H205K mutants exhibited only feeble activity, while the H310A, H310K, and D52A mutants were totally inactive, indicating that these conserved residues, particularly Asp52 and His310, were essential in the catalytic mechanism. Furthermore, bioinformatic analysis revealed that GAG sulfatases with specific degradative properties clustered together in the neighbor-joining phylogenetic tree. Based on this finding, 60 Δ4,5hexuronate-2-O-sulfatases were predicted in the NCBI protein database, and one with relatively low identity to PB2SF was characterized to confirm our prediction. Moreover, the signature sequences of bacterial Δ4,5hexuronate-2-O-sulfatases were identified. With the reported signature motifs, the sulfatase sequence of the Δ4,5hexuronate-2-O-sulfatase family could be simply identified before cloning. Taken together, the results of this study should aid in the identification and further application of novel GAG sulfatases

The application of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method in microbial screening

A rapid microbial screening method was developed based on denaturing gradient gel electrophoresis (DGGE). To evaluate the repeatability and reliability of this system, three DGGE markers were used. Then, the feasibility of DGGE method was verified by microbial screenings in viili (a traditional fermented dairy product originated from Scandinavia) and sourdoughs. The results suggested that, this method could efficiently classify the duplicate strains isolated from the complex environment and identify their dominance in microbial ecology if the corresponding environment samples had been provided. This paper proposed the application of polymerase chain reaction (PCR)-DGGE method in reducing the complicated work in microorganism identifications or even directly identified the target strains if a proper DGGE marker had been applied.Key words: Denaturing gradient gel electrophoresis (DGGE), microbial screening, bacteria, fungi, Lactobacillus, bifidobacteria, sourdoughs, viili