Bacterial Porin in the Ocean and Ecological Implications

Mortality of Microbes in Aquatic Environments

Microbial Biosystems: New Frontiers

Proceedings of the 8

International Symposium on Microbial Ecology

Bell CR, Brylinsky M, Johnson-Green P (eds)

Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999.

Bacterial porin in the ocean and its ecological implications

S. Suzuki

, K. Kogure

, E. Tanoue

Department of Aquaculture, Kochi University, Kochi 783-8502, Japan

Ocean Research Institute, University of Tokyo, Tokyo 164-8639, Japan

Institute for Hydrospheric-Atmospheric Sciences, Nagoya University, Nagoya 464-8601, Japan

ABSTRACT

Dissolved protein is an important component of the flow of organic substances in the sea.

This study attempted to elucidate the mechanisms responsible for the formation and

stabilization of specific dissolved protein molecules. The dissolved proteins studied were

the porin homologues of Pseudomonas and Vibrio. A survey using antisera specific for

bacterial cell (anti-P. aeruginosa) and porin P that most likely originated from P.

aeruginosa (anti-48DP N-14) revealed that cell numbers detected with anti-48DP N-14

were higher than that detected with anti-P. aeruginosa. This suggests that porin

homologues of P. aeruginosa are present in bacteria other than P. aeruginosa.

Furthermore, antiserums against porin of V. anguillarum (anti-Omp35La), which can

detect major porin of Vibrio genera, reacted similar to proteins of non-Vibrio group

bacteria. These results strongly suggest that the porins originated from a diversity of

bacterial groups rather than solely from the two genera above, and that they are able to

survive and form dissolved proteins in the ocean.

Introduction

Dissolved organic matter (DOM) in the ocean is one of the largest pools of organic matter

on the planet. DOM includes a diversity of molecules including proteins, nucleic acids,

polysaccharides and numerous low molecular weight compounds. These biological

molecules are believed to be largely decomposed into inorganic matter, although some

become geochemicaly transformed into polymerized organic compounds that are relatively

resistant to degradation.

In recent years, however, it was found that 48 kDa specific dissolved protein is present

as DOM and does not appear to be degraded [1]. Fig. 1 represents a hypothetical

illustration of the fate of these specific proteins in relation to the overall degradation of

biological materials. N-terminal amino acid analysis of the 48 kDa protein revealed that

the dissolved protein is a homologue of a bacterial porin OprP of Pseudomonas aeruginosa

[2]. The OprP was induced when P. aeruginosa was exposed to phosphate deficient

environments. However, P. aeruginosa is a human pathogen and is not found in the ocean.

These findings lead to the questions of what the 48 kDa OprP homologue is and why is it

present in the ocean. In an attempt to answer these questions we carried out studies using

antibody probes against two porins to determine whether there are dissolved proteins

homologous to bacterial porins in seawater samples collected in subarctic, subtropical,

tropical and antarctic areas. We also carried out trials to directly count and isolate probe-

reactive bacteria. We here summarize the discovery of the porin homologues in seawater

and some more recent findings.

Mortality of Microbes in Aquatic Environments

Fig. 1 Metabolic pathway of biomolecules in the ocean. The specific dissolved proteins escape from

degradation. A new pool is added to the pathway proposed by Cauwet [1].

Dissolved Proteins in the Ocean

Tanoue et al. [5] discovered that approximately 30 polypeptides could be detected in SDS-

PAGE in concentrated seawater samples that were collected from the arctic to the antarctic.

Among the polypeptides, the 48 kDa-band was found in most samples suggesting that this

protein is widely distributed in the ocean. The N-terminal amino acid sequence revealed

that the 48 kDa protein has a 100% identical sequence in the N-terminus to that of

Pseudomonas aeruginosa

porin OprP. Since

P. aeruginosa

has not been recognized as

being present in the ocean, it was questioned as to what the origin of the 48 kDa protein is.

In order to examine the distribution of the 48 kDa protein molecule in many samples, an

antibody against the primary sequence of the N-terminal amino acids of the protein was

prepared [4]. Using this immunological probe (anti-48DP N-14), it was found that the

dissolved proteins have similar antigenicity to the N-terminal 14mer of the 48 kDa protein

[4]. We also used another antibody which recognizes the Omp35La porin of

Vibrio

anguillarum

and other species belonging to the genus

Vibrio

[3]. This antibody reacted to

34 to 48 kDa proteins of seawater samples, indicating that the porin of the

Vibrio

genus is

also a candidate for the origin of the dissolved protein [4]. The above suggests that

bacterial porins may be one of the sources of the dissolved protein.

Bacteria Producing Dissolved Proteins

Studies were carried out to determine what kinds and how many bacteria produce the porin

homologues reacting to the antisera. The first experiment was conducted by the University

of Tokyo group. Using immunofluorescent microscopy, they counted the bacteria reactive

to anti-

P. aeruginosa

whole cell antibody (commercially available anti-

P. aeruginosa

) and

to anti-48DP N-14 in a sample collected during the Tnasei-maru R/V (KT-97-6) research

cruise. They found the number of cells reactive to anti-48DP N-14 to be much greater than

the number reactive to anti-

P. aeruginosa

(Kimata, unpublished data). This suggests that

bacteria possessing antigen similar to

P. aeruginosa

are present in the ocean. Furthermore,

it was found that bacteria other than the anti-

P. aeruginosa

reactive cells possess similar

Mortality of Microbes in Aquatic Environments

antigenic sites to the N-terminal 14mer of the 48 kDa dissolved protein and

P. aeruginosa

OprP. These results indicate that the origin of the OprP homologue is not from one

species.

The second experiment was carried out by the Kochi University group using anti-

Omp35La. Culturable bacteria were isolated from the sample obtained on the KT-97-6

cruise. Anti-Omp35La reactive bacteria were screened by colony-Western blotting. After

screening two times, 37 isolates were recognized as real positives and these were used for

further Western blotting using their outer membrane proteins (OMP). The bacteria having

reactive proteins sizing 30 to 40 kDa composed 16 strains. Other strains were negative in

Western blotting suggesting that their positive reaction in colony-Western blotting was due

to other antigenic substances, such as lipopolysaccharides. The strains with anti-Omp35La

reactive OMP were classified by general biochemical properties and restriction fragment

length polymorphism (RFLP) analysis. The antibody reactive isolates included species of

the

Vibrio

genus and other genera. RFLP analysis showed that all Omp35La producing-

isolates were not

V. anguillarum

alone, although strains close to

V. orientralis

pelagium

, and

V. tubiasii

were included. These results indicate that the Omp35La

homologue was produced by other organisms beside

V. anguillarum

. Taken together, this

strongly suggests that the organisms responsible for producing the homologue are not

restricted to the genus

Pseudomonas

and

Vibrio

Stability of Omp35La Against Protease

In order to examine the stability of bacterial porin in the ocean, we designed a preliminary

in vitro

experiment to test whether Omp35La is hydrolyzed by protease. Since it was

hypothesized that stability is dependent on the physical form of the protein, the outer

membrane containing Omp35La and isolated-Omp35La were prepared from

anguillarum

, and then digested with trypsin. The results showed that Omp35La embedded

in the membrane envelope was resistant to trypsin. When analyzed by native SDS-PAGE

without using a reducing agent or heating, Omp35La was detected as trimer, indicating that

this protein is present as native trimer after treatment with trypsin. Other minor proteins

associated with the envelope were hydrolyzed under this condition. On the other hand,

isolated Omp35La was hydrolyzed with trypsin, although Omp35La was more stable than

other proteins. This suggests that if in the natural environment the porins are present

embedded in membrane, the proteins may be protected from protelytic degradation. A

schematic drawing of the possible stabilizing mechanism of the porins and related

membrane proteins is presented in Fig. 2. We plan to carry out further experiments to

clarify the mechanism of the stability of the dissolved protein molecules. Bacterial cells

are decomposed by many kinds of processes, such as infection by phage and by grazing by

heterotrophic nanoflagellates. The different "cell death" processes are thought to be

important in determining the nature of the specific protein pool.

Conclusions and Unanswered Questions

1. The source of dissolved proteins in sea water is partly from bacterial porins and

possibly from related membrane proteins.

2. Porin embedded in membrane is resistant to proteolytic degradation.

3. The native form of the dissolved protein is not known.

4. The quantity and dynamics of dissolved protein in the ocean is not well understood.

Overview

Bacterial Porin in the Ocean and Ecological Implications

Bacterial porins play a crucial role in the ocean's ecosystem by contributing to the pool of dissolved organic matter. This research explores the presence and stability of porin homologues from Pseudomonas and Vibrio species in marine environments. The findings indicate that these proteins can survive degradation and are produced by a diverse range of bacteria. Understanding the mechanisms behind dissolved proteins in seawater is essential for comprehending nutrient cycling and microbial ecology. This study is relevant for marine biologists and ecologists interested in microbial interactions and organic matter dynamics. Key Points Investigates the role of bacterial porins in oceanic dissolved organic matter. Examines the stability of porin proteins against degradation in marine …

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FAQs

What are bacterial porins and their significance in the ocean?

Bacterial porins are proteins that facilitate the transport of molecules across bacterial membranes. In the ocean, they contribute significantly to the pool of dissolved organic matter, which is crucial for nutrient cycling and microbial ecology. This study highlights the presence of porin homologues from species like Pseudomonas and Vibrio, suggesting that they play a vital role in marine ecosystems. Understanding these proteins helps researchers grasp the dynamics of organic matter in oceanic environments.

How do porin proteins survive degradation in seawater?

The research indicates that porin proteins, particularly those embedded in bacterial membranes, exhibit resistance to proteolytic degradation. When tested in vitro, porins like Omp35La from Vibrio anguillarum were found to remain stable when associated with their membranes. This stability suggests that in natural environments, porins can persist longer, contributing to the dissolved protein pool in oceans. The findings emphasize the importance of understanding protein stability for ecological studies.

What methods were used to identify porin homologues in seawater?

The study utilized immunological probes, specifically antibodies against known porins, to detect homologues in seawater samples collected from various marine environments. Techniques such as immunofluorescent microscopy and colony-Western blotting were employed to identify and quantify bacteria producing these proteins. The results revealed a diversity of bacterial sources, indicating that porins are not exclusive to Pseudomonas and Vibrio, but are produced by a range of marine bacteria.

What ecological implications arise from the presence of dissolved proteins in the ocean?

Dissolved proteins, particularly those derived from bacterial porins, have significant ecological implications for nutrient cycling in marine environments. They contribute to the organic matter pool, which supports various microbial processes and food webs. The persistence of these proteins can influence the availability of nutrients for other organisms, thereby affecting overall marine productivity. Understanding these dynamics is crucial for marine ecology and conservation efforts.

What are the key findings regarding the distribution of porin proteins in marine environments?

The study found that the 48 kDa porin homologues are widely distributed across different marine environments, from subarctic to tropical waters. This suggests that these proteins are not limited to specific bacterial species but are produced by a variety of marine bacteria. The presence of these proteins in diverse locations indicates their ecological importance and potential role in global biogeochemical cycles. These findings highlight the need for further research into the sources and functions of dissolved proteins in the ocean.