Utilization of Mucus from the Coral Acropora palmata by the Pathogen Serratia marcescens and by Environmental and Coral Commensal Bacteria

In recent years, diseases of corals caused by opportunistic pathogens have become widespread. How opportunistic pathogens establish on coral surfaces, interact with native microbiota, and cause disease is not yet clear. This study compared the utilization of coral mucus by coral-associated commensal bacteria (“Photobacterium mandapamensis” and Halomonas meridiana) and by opportunistic Serratia marcescens pathogens. S. marcescens PDL100 (a pathogen associated with white pox disease of Acroporid corals) grew to higher population densities on components of mucus from the host coral. In an in vitro coculture on mucus from Acropora palmata, S. marcescens PDL100 isolates outgrew coral isolates. The white pox pathogen did not differ from other bacteria in growth on mucus from a nonhost coral, Montastraea faveolata. The ability of S. marcescens to cause disease in acroporid corals may be due, at least in part, to the ability of strain PDL100 to build to higher population numbers within the mucus surface layer of its acroporid host. During growth on mucus from A. palmata, similar glycosidase activities were present in coral commensal bacteria, in S. marcescens PDL100, and in environmental and human isolates of S. marcescens. The temporal regulation of these activities during growth on mucus, however, was distinct in the isolates. During early stages of growth on mucus, enzymatic activities in S. marcescens PDL100 were most similar to those in coral commensals. After overnight incubation on mucus, enzymatic activities in a white pox pathogen were most similar to those in pathogenic Serratia strains isolated from human mucosal surfaces.Serratia is a gammaproteobacterium frequently isolated from waters, plants, and animals (7). Some isolates of Serratia are well-characterized symbionts of invertebrates. Serratia marcescens and Serratia liquefaciens have been identified as vertically transmitted symbionts of the sugar beet maggot (9). Serratia colonizes male and female reproductive tracts of the maggots, eggs, and pharyngeal filter. There, the bacteria are hypothesized to aid in metamorphosis by digesting chitinous puparial walls (9). In the gut of another insect, the diamondback moth, strains of S. marcescens appear to live as commensals capable of modestly (5 to 8%) increasing growth rates of the host (8). Serratia strains have also been isolated from feces and cloacal swabs from clinically normal captive birds, but not from organs or carcasses of sick or diseased animals housed within the same facility (3, 20). Serratia spp. have also been linked to diseases of invertebrate animals and their larvae (for reviews, see references 7, 15, and 21). To cause diseases in nematodes and flies, S. marcescens first colonizes the intestines, degrades cells of the alimentary tract and then spreads to other organs (14, 21). There are, however, exceptions to this mode of infection. Serratia entomophila, the causal agent of amber disease in grubs, grows within the alimentary tract of the animal to >10⁶ CFU. However, bacteria neither attach to nor colonize surfaces of the gut; rather, they adhere to gut contents (10) and cause the appearance of signs by producing the Sep toxin that inhibits accumulation of the insect''s digestive serine proteases and disrupts the cytoskeletal network (6). It appears, therefore, that various isolates of Serratia are capable of entering into a full range of interactions (from mutualistic to commensal to pathogenic) with their animal hosts (for reviews, see references 7, 15, and 21).A strain of S. marcescens, PDL100, was shown to be associated with white pox disease of the threatened Caribbean coral Acropora palmata (22, 27). White pox disease results in coral tissue necrosis, exposing carbonate skeleton at a rate of 2.5 cm² day⁻¹ (22). It is not yet clear how S. marcescens PDL100 colonizes and infects corals. It is likely that to cause disease, the pathogen first needs to colonize and establish within the coral surface mucus layer.The coral surface mucus layer contains polymers of mixed origin. Coral mucus is made in the mucocytes of the polyp, where the photosynthate produced by the coral symbiotic dinoflagellate Symbiodinium spp. is converted into polymers that are excreted onto the coral surface (for a review, see reference 2). A glycoprotein is the major component of coral mucus from both hard and soft corals (16, 17, 19). The composition of the glycoprotein differs among coral species (4, 17). The mucus polymer of Acropora formosa, for example, contains 36 to 38% of neutral sugars, 18 to 22% of amino sugars, and 19 to 30% of amino acids; lipids make up 4.2% of the polymer (17). In the mucus of A. formosa, the oligosaccharide decorations (two to four sugar residues long) are attached to the polypeptide backbone by an O-glycosidic link to serine or threonine through the carbon 1 of mannose (16). The glycoproteins from A. formosa and Pseudopterogorgia americana corals contain terminal arabinose residues linked by a β1→2 or β1→3 bond. In the mucus of acroporid corals, arabinose, N-acetyl-glucosamine, mannose, glucose, galactose, N-acetyl-galactosamine, and fucose were the major sugars; serine and threonine were the major amino acids (4, 17). The elucidation of the chemical structure of coral mucus is complicated by the fact that the mucus contains excretions of coral mucocytes, extracellular substances produced by the associated microbiota as well as oligomers that may result from the degradation of these polymers (for reviews, see references 2 and 24).In this study, we tested the hypothesis that S. marcescens PDL100 is capable of a more extensive utilization of A. palmata mucus than other environmental or pathogenic isolates of S. marcescens. This hypothesis is based on the recent discoveries that pathogenic and commensal host-associated bacteria differ in their patterns of carbon source utilization during growth on components of the mucus that lines host surfaces (5, 26). These different strategies of mucus utilization may allow pathogenic bacteria to outcompete native residents and establish within the host''s mucosa (5, 13, 26). To test this hypothesis, growth of the strain PDL100 on coral mucus and enzymatic activities induced during growth on mucus were assayed and compared to those of pathogenic and environmental isolates of S. marcescens and three native coral-associated bacteria.     

Structurally complex habitats provided by Acropora palmata influence ecosystem processes on a reef in the Florida Keys National Marine Sanctuary

The disappearance of Acropora palmata from reefs in the Florida Keys National Marine Sanctuary (FKNMS) represents a significant loss in the amount of structurally complex habitat available for reef-associated species. The consequences of such a widespread loss of complex structure on ecosystem processes are still unclear. We sought to determine whether the disappearance of complex structure has adversely affected grazing and invertebrate predation rates on a shallow reef in the FKNMS. Surprisingly, we found grazing rates and invertebrate predation rates were lower in the structurally complex A. palmata branches than on the topographically simple degraded reefs. We attribute these results to high densities of aggressively territorial damselfish, Stegastes planifrons, living within A. palmata. Our study suggests the presence of agonistic damselfish can cause the realized spatial patterns of ecosystem processes to deviate from the expected patterns. Reef ecologists must therefore carefully consider the assemblage of associate fish communities when assessing how the mortality of A. palmata has affected coral reef ecosystem processes.     

Recruitment failure in Florida Keys <Emphasis Type="Italic">Acropora palmata,</Emphasis> a threatened Caribbean coral

Recovery of Acropora palmata from its currently imperiled status depends on recruitment, a process which is poorly documented in existing Caribbean coral population studies. A. palmata is thought to be well adapted to proliferate through the recruitment of fragments resulting from physical disturbances, such as moderate intensity hurricanes. This study monitored fifteen 150 m² fixed study plots on the upper Florida Keys fore-reef for asexual and sexual recruitment from 2004 to 2007. Between July and October 2005, 4 hurricanes passed by the Florida Keys, producing wind speeds on the reef tract of 23 to 33 m s⁻¹. Surveys following the hurricanes documented an average loss of 52% estimated live tissue area within the study plots. The percentage of “branching” colonies in the population decreased from 67% to 42% while “remnant” colonies (isolated patches of tissue on standing skeleton) increased from 11% to 27%. Although some detached branches remained as loose fragments, more than 70% of the 380 fragments observed in the study plots were dead or rapidly losing tissue 3 weeks after Hurricane Dennis. Over the course of the study, only 27 fragments became attached to the substrate to form successful asexual recruits. Meanwhile, of the 18 new, small encrusting colonies that were observed in the study, only 2 were not attributable to asexual origin (i.e., remnant tissue from colonies or fragments previously observed) and are therefore possible sexual recruits. In summary, the 2005 hurricane season resulted in substantial loss of A. palmata from the upper Florida Keys fore-reef from a combination of physical removal and subsequent disease-like tissue mortality, and yielded few recruits of either sexual or asexual origin. Furthermore, the asexual and sexual fecundity of the remaining population is compromised for the near future due to the lack of branches (i.e., “asexual fecundity”) and overall loss of live tissue.     

Barium variation in Acropora palmata and Montastrea annularis

The barium contents of Acropora palmata and Montastrea annularis are distinct both in their averages (6 and 9 ppm, respectively) and, more impressively, in their variability (5 to 7 ppm and 6 to 15 ppm). A. palmata contained about 50% more Ba²⁺ than M. annularis and exhibited more than three times as much variation, as measured by their respective coefficients of variation. In contrast, the means and coefficients of variation of Sr²⁺ in these groups differed by only 6% and 15%. No obvious environmental or post-depositional causes for the inter-and intra-specific variation of Ba²⁺ could be found. Previous experiments indicate that the partitioning of Ba²⁺ into aragonite depends directly on the rate of precipitation. This suggests that Ba²⁺ does not substitute for Ca²⁺ but instead is incorporated by occlusion. Since growth rates in A. palmata often exceed those of M. annularis, this appears to be the mechanism which generates higher and more variable concentrations of Ba²⁺ in A. palmata.     

Assessment of Acropora palmata in the Mesoamerican Reef System

The once-dominant shallow reef-building coral Acropora palmata has suffered drastic geographical declines in the wider Caribbean from a disease epidemic that began in the late 1970s. At present there is a lack of quantitative data to determine whether this species is recovering over large spatial scales. Here, we use quantitative surveys conducted in 107 shallow-water reef sites between 2010 and 2012 to investigate the current distribution and abundance of A. palmata along the Mesoamerican Reef System (MRS). Using historical data we also explored how the distribution and abundance of this species has changed in the northern portion of the MRS between 1985 and 2010–2012. A. palmata was recorded in only a fifth of the surveyed reef sites in 2010–2012. In the majority of these reef sites the presence of A. palmata was patchy and rare. Only one site (Limones reef), in the northernmost portion of the MRS, presented considerably high A. palmata cover (mean: 34.7%, SD: 24.5%). At this site, the size-frequency distribution of A. palmata colonies was skewed towards small colony sizes; 84% of the colonies were healthy, however disease prevalence increased with colony size. A comparison with historical data showed that in the northern portion of the MRS, in 1985, A. palmata occurred in 74% of the 31 surveyed sites and had a mean cover of 7.7% (SD = 9.0), whereas in 2010–2012 this species was recorded in 48% of the sites with a mean cover of 2.9% (SD = 7.5). A. palmata populations along the MRS are failing to recover the distribution and abundance they had prior to the 1980s. Investigating the biological (e.g., population genetics) and environmental conditions (e.g., sources of stress) of the few standing reefs with relatively high A. palmata cover is crucial for the development of informed restoration models for this species.     

Role of Methionine in Biosynthesis of Prodigiosin by Serratia marcescens

Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratia marcescens strain Nima. However, when methionine was added to NPC synthesizing prodigiosin in the presence of other amino acids, the lag period for synthesis of prodigiosin was shortened, an increased amount of the pigment was formed, and the optimal concentrations of the other amino acids were reduced. Less prodigiosin was synthesized when addition of methionine was delayed beyond 4 h. The specific activity of prodigiosin synthesized by addition of (14)CH(3)-methionine was 40 to 50 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. NPC of mutant OF of S. marcescens synthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and the specific activity of this pigment synthesized in the presence of (14)CH(3)-methionine was only 5 to 13 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. A particulate, cell-free extract of mutant WF of S. marcescens methylated norprodigiosin to form prodigiosin. When the extract was added to NPC of mutant OF synthesizing norprodigiosin in the presence of (14)CH(3)-methionine, the prodigiosin formed had 80% greater specific activity than the norprodigiosin synthesized in the absence of the extract. The C6 hydroxyl group of norprodigiosin was methylated in the presence of the extract and methionine. Biosynthesis of prodigiosin by NPC of strain Nima also was augmented by addition of S-adenosylmethionine. Various analogues of methionine such as norleucine, norvaline, ethionine, and alpha-methylmethionine did not affect biosynthesis of prodigiosin by NPC either in the presence or absence of methionine.     

Cation transport in Serratia marcescens and Serratia marinorubra

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba, were determined by atomic-absorption spectrometry. The intracellular K(+) and Mg(2+) contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg(2+) content of S. marinoruba was generally greater than that of S. marcescens. The Na(+) content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K(+) by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra. The slower rate of K(+) transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K(+) transport was found to have a pH optimum of 5.5 to 6.1 for S. marcescens, and the K(m) for K(+) was approximately 1.6 mm. Na(+) and Mg(2+) were not taken up by the cells, although the presence of Mg(2+) tended to decrease rates of K(+) uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at pH >7.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Role of the outer membrane in the accumulation of quinolones by Serratia marcescens

Accumulation of four quinolones by Serratia marcescens was measured fluorometrically. The passage of quinolones through the outer membrane was studied in both lipopolysaccharide-deficient and porin-deficient mutants. The lipopolysaccharide (LPS) layer formed a partially effective barrier for highly hydrophobic quinolones such as nalidixic acid. Quinolones with a low relative hydrophobicity coefficient seemed to pass preferentially through the water-filled Omp3 porin channels. Results were confirmed when Omp3 was cloned in a porin-defective Escherichia coli.     

X-Ray Analysis of the Magnesium-containing Endonuclease from Serratia marcescens

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescenswas refined at the resolution of 1.07 Å to Rfactor of 12.4% and R _freefactor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg²⁺ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N-and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg²⁺ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O¹atom of Asn119 were shown to be within the range of 2.01–2.11 Å. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 Ų, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 Ų, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

Staghorn tempestites in the Florida Keys

Thirty-one samples of transported Holocene Acropora cervicornis "sticks" sampled from carbonate sand tempestite accumulations at 19 sites along a 180-km-long stretch of the Florida reef tract were dated using the radiocarbon (¹⁴C) method. The "modern fossils" collected from just a few centimeters below the surface ranged in age from 0.5 to 6.4 ka. The majority lived between 3.5 and 5.5 ka. The time of transport and deposition is not known. There were no A. cervicornis samples centered around 4.5 ka. Acropora cervicornis is living on many Florida reefs, but the youngest tempestite sample was 500 years old. Two 500-year-long gaps in dated staghorn suggest that the documented decline in living A. cervicornis over the past 25 years may not be without precedent.     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.     

粘质沙雷氏菌几丁质酶chiB基因的克隆与序列分析

采用改进的方法提取粘质沙雷氏菌基因组DNA,通过PCR扩增,得到大小为1 500 bp特异性DNA片段(chiB基因),以pUC18质粒构建了pUC-ch iB克隆载体,转化至感受态细胞E.coliDH5α培养,并筛选出重组质粒。经测序分析,证明克隆片段与文献报道相一致。     

Transductional Analysis of the leu-thr Region of Chromosome of Serratia marcescens

Transductional analysis of the chromosomal linkage map of Serratia marcescens near leu revealed the following order of loci: ser4-thr3-pyr1-pdx2-leu1-azi8.