Sequence Analysis of a 101-Kilobase Plasmid Required for Agar Degradation by aMicroscillaIsolate

Abstract

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.Bacteria have long been exploited as a source of natural products for use in medicine, agriculture, and industry. Until recently, most studies utilized microbes that had been isolated from clinical or terrestrial environments. It is now recognized that bacteria living in marine environments provide an abundant and as-yet-untapped source of metabolic properties (8). Marine sediments are rich in bacteria, with reports of 10 8 to 10 10 microbes per g of sediment for the first few centimeters of sediment (21). Estimates of bacterial diversity in natural environments have indicated that, while a few organisms may predominate, such environments still represent a highly complex assemblage of microbes (45).We have initiated a study to identify and characterize the genes present on plasmids isolated from bacteria present in coastal marine sediments. Extrachromosomal elements by definition encode functions that are not essential for cell growth but which provide an advantage to the host bacterium under certain growth conditions. It is therefore not surprising that a wide variety of traits in bacteria have been found to be plasmid encoded. In an earlier study (41), ca. 30% of more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments contained at least one plasmid that ranged in size from 5 to Ͼ250 kb. These plasmids appeared to contain novel and generally uncharacterized replication regions since no homology was detected between ca. 300 plasmids of marine origin (41) and 15 replicon probes derived from plasmids found in bacteria isolated from mammalian or terrestrial sources (10). These findings suggested that plasmids in marine sediment microbial communities are a unique and diversified set of extrachromosomal elements.We present here the characterization of an agar-degrading marine isolate, Microscilla sp. strain PRE1. This organism contains a 101-kb plasmid, designated pSD15, which potentially encodes five different agarases and is essential for the ability of the bacterium to degrade agar. The complete DNA sequence and analysis of pSD15 are reported. MATERIALS AND METHODSIsolation and characterization of marine bacterial isolate PRE1. Marine sediment associated with the roots of pickleweed (Salicornia virginica) was collected from the Kendall-Frost Mission Bay Marsh Reserve in San Diego, Calif. The sediment was resuspended in artificial seawater (0.3 M NaCl, 0.1 M KCl,...

Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes

“…The acquisition of the B. plebeius porphyran PUL is likely to have originated from a marine Bacteroidete because similar gene clusters are present in oceanic species (8,(30)(31)(32). Because the B. plebeius PUL is harbored at a similar location on an otherwise very similar ICE that is present in B. thetaiotaomicron, this finding suggests that HGT via conjugative elements is the mechanism of transfer and that similar ICE vehicles are capable of acquiring new cargo (20).…”

Enzymatic Properties and Nucleotide and Amino Acid Sequences of a Thermostable β-Agarase from the Novel Marine Isolate, JAMB-A94

“…The pH optimum for activity of rAgaA (0.02 U/ml) was examined at 50 C in 50 mM Britton-Robinson universal buffers (pH [3][4][5][6][7][8][9][10][11][12]. The enzyme showed an optimal pH of about 7.0.…”

“…2) Agar-degrading bacteria isolated from marine and other environments so far have been assigned to the genera Pseudomonas, 3) Pseudoalteromonas, 4) Streptomyces, 5) Alteromonas, 6) Microscilla, 7,8) Vibrio, 9,10) and Cytophaga.…”

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

“…They are grouped into ␣ and ␤ types that hydrolyze ␣-1,3 linkages and ␤-1,4 linkages, respectively (4). Agarases have been isolated from several bacteria genera, including Cytophaga (5), Pseudomonas (6 -9), Pseudoalteromonas (10), Zobellia (11), Streptomyces (12), Alteromonas (13), Vibrio (14,15), Microscilla (16), Microbulbifer (17), and Aeromonas. Most known agarases are ␤-agarases, with the exception of two ␣-agarases found from Alteromonas agarlyticus GJ1B (18,19) and Thalassomonas sp.…”