Occurrence and degradation of peptidoglycan in aquatic environments

FEMS Microbiology Ecology 46 (2003) 269^280

www.fems-microbiology.org

Occurrence and degradation of peptidoglycan in aquatic environments Niels O.G. Jrgensen a

a;


, Ramunas Stepanaukas b;1 , Anne-Grethe U. Pedersen c , Michael Hansen a , Ole Nybroe a

Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Ecology/Limnology, Lund University, S-22362 Lund, Sweden c Department of Microbial Ecology, University of Aarhus, Ny Munkegade, DK-8000 Aarhus C, Denmark Received 1 December 2002; received in revised form 21 March 2003 ; accepted 26 April 2003 First published online 27 August 2003

Abstract Mechanisms controlling microbial degradation of dissolved organic matter (DOM) in aquatic environments are poorly understood, although microbes are crucial to global nutrient cycling. Bacterial cell wall components may be one of the keys in understanding the presence of slowly degrading DOM in nature. We found that dominant components of bacterial cell walls (D-amino acids (D-AA), glucosamine (GluA) and diaminopimelic acid (DAPA)) comprised up to 11.4% of the dissolved organic nitrogen in 50 diverse rivers entering the Baltic Sea. Occurrence of DAPA, a characteristic component of Gram-negative (G3 ) bacteria, in the rivers suggests that G3 bacteria rather than Gram-positive (Gþ ) were the major source of the cell wall material. In laboratory studies, the degradation of whole bacterial cells, cell wall material and purified peptidoglycan was studied to characterize degradation of cell wall material by natural aquatic bacteria. Addition of whole killed G3 and Gþ bacteria to cultures of estuarine bacteria demonstrated fragmentation and loss of cell structure of the Gþ bacteria, while the G3 bacteria maintained an intact cell shape during the entire 69-day period. In another experiment, estuarine bacteria degraded 39^69% of GluA, D-AA and DAPA in added cell wall material of a representative G3 bacterial species during 8 days, as compared to a 72^89% degradation of GluA, D-AA and DAPA in cell material of a Gþ bacterial species. When cultures of estuarine bacteria were enriched with purified G3 and Gþ peptidoglycan (1 mg l31 ), at least 49% (G3 ) and 58% (Gþ ) of D-AA in the peptidoglycan was degraded. No major changes in GluA were obvious. Interpretation of the results was difficult as a portion of the purified peptidoglycan was of similar size to the bacteria and could not be differentiated from cells growing in the cultures. Addition of the purified peptidoglycan stimulated the bacterial growth, and after 6 days the cell density in the enriched cultures was 4-fold higher than in the controls. A regrowth of bacteria after addition of L-broth at 105 days caused a 50- to 75-fold increase in dissolved D-AA and GluA. Most of the D-AA and GluA were taken up during the following 10 days, indicating that cell wall constituents are dynamic compounds. Our results show that a variable portion of peptidoglycan in G3 and Gþ bacteria can be degraded by natural bacteria, and that peptidoglycan in G3 bacteria is more resistant to bacterial attack than that in Gþ bacteria. Thus, the presence of cell wall constituents in natural DOM may reflect the recalcitrant nature of especially G3 peptidoglycan. @ 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Peptidoglycan ; Bacterial cell wall ; Baltic river; Degradation ;

D-amino

1. Introduction An important function of bacteria in nature is the degradation of dissolved organic matter (DOM), but bacteria themselves also produce DOM components. Certain bac-

* Corresponding author. Tel. : +45-3528-2625; Fax: +45-3528-2606. E-mail address : [email protected] (N.O.G. Jrgensen). 1

Present address: Savannah River Ecology Laboratory, Aiken, SC 29803, USA.

acid ; Glucosamine ; Diaminopimelic acid

terial products have been found recalcitrant to microbial decomposition, including membrane proteins [1], liposome-like particles formed after protozoan grazing [2], and unidenti¢ed organic molecules rich in sugars and amino acids (AA) [3]. In addition, bacterial cell wall components have been found to constitute a major fraction of recalcitrant DOM in the ocean [4]. The cell wall of Gram-positive (Gþ ) bacteria consists of a thick and uniform peptidoglycan layer, forming 90% of the cell wall. In contrast, Gram-negative (G3 ) bacteria have a complex, multilayered cell wall structure with a

0168-6496 / 03 / $22.00 @ 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00194-6

FEMSEC 1555 24-11-03

Cyaan Magenta Geel Zwart

270

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

relatively thin inner peptidoglycan layer (constitutes 10% of the cell wall) and an outer membrane of lipopolysaccharides and proteins [5]. Peptidoglycan consists of strands of alternating L-1,4-linked N-acetylglucosamine and N-acetylmuramic acid units, cross-linked by short peptides [5]. AA in these peptides include D-isomers of alanine, aspartate, glutamate, and serine, which have been used as markers of peptidoglycan in environmental studies. In marine waters and sediments, D-AA have been found to make up 10^30% of the hydrolyzable AA, indicating that peptidoglycan may comprise a relatively large fraction of DOM [4,6,7]. Considering that 6 10% of all AA in living marine bacteria are D-isomers (unpublished), and that bacterial biomass in pelagic waters and sediments makes up 6 5% of the total organic carbon pool2 , a proportion of D-isomers s 10% in this organic matter indicates a 20- to 60-fold enrichment with D-AA. Most likely, this increase is caused by a selective biological degradation of L-AA relative to D-AA. The enrichment of DOM with AA that are speci¢c or typical to bacterial cell walls may indicate a reduced degradation of cell wall components, but the enhanced concentrations do not provide information on the mechanisms controlling the degradation of, e.g. peptidoglycan. To shed light on relations between occurrence of cell wall AA and degradation of peptidoglycan, we surveyed occurrence of various peptidoglycan components in 50 rivers discharging into the Baltic Sea and studied degradation of cell wall components by estuarine bacteria. The Baltic rivers represent diverse freshwater ecosystems on a broad geographical scale [8]. The rivers were analyzed for content of glucosamine (GluA), diaminopimelic acid (DAPA), and D-isomers of alanine, aspartate, glutamate, and serine. DAPA is a peptide component of peptidoglycan in G3 bacteria but is typically replaced by lysine in Gþ bacteria [5]. Microbial degradation of bacterial cell wall material was analyzed in laboratory cultures of growing estuarine bacteria that were amended with whole ultraviolet (UV)-killed bacteria, crude cell material or puri¢ed peptidoglycan. Cell walls of both Gþ and G3 bacteria were included in the experiments due to the di¡erence in structure and composition of their cell walls. These di¡erences may cause a preferential degradation of intact cell walls and speci¢c cell wall components in Gþ relative to G3 bacteria. This was supported by our results, which indicated that both whole

cells, cell walls and peptidoglycan were more resistant to microbial degradation in G3 than in Gþ bacteria.

2. Materials and methods 2.1. Sampling and analysis of nitrogen compounds Water samples were collected in July 1999 at the mouth of 50 rivers discharging into the Baltic Sea. The drainage basin of the Baltic Sea includes intensive agriculture at the southern part, while the northern part is almost entirely covered by boreal forest [8]. The water samples were ¢ltered through 0.45-Wm ¢lters to remove particulate material. Details on sampling, water chemistry and concentrations of nutrients are given in Stepanaukas et al. [8]. Dissolved organic nitrogen (DON) was measured as total dissolved nitrogen by chemiluminescence after conversion to NOx and subtraction of dissolved inorganic nitrogen (ammonium and nitrate) [9]. Water samples for analysis of dissolved D-AA (D-Asp, D-Glu, D-Ser and D-Ala), GluA and DAPA were hydrolyzed by a microwave-assisted vapor-phase technique [10] before high performance liquid chromatography (HPLC) analysis. For quanti¢cation of D-AA and GluA, the hydrolyzed material was derivatized with o-phthaldialdehyde and N-isobutyrylL-cysteine as a chiral agent [11] and separated on a Waters XTerra RP18 3.5-Wm particle column (Waters Corporation, Milford, USA) at a £ow rate of 0.7 ml min31 . Mobile phases were (A) aqueous solution of 5 mM Na2 H2 PO4 , 45 mM sodium acetate trihydrate and 7.5% methanol at pH 6.4, and (B) 100% methanol [12]. The derivatives were eluted with the following gradient: T0 min (100% A, 0% B), T27 min (50% A, 50% B), T30 min (10% A, 90% B) and T33 min (100% A, 0% B). The o-phthaldialdehyde and N-isobutyryl-L-cysteine mixture forms £uorescent derivatives with GluA but not with N-acetylglucosamine. But since the hydrolysis converted N-acetylglucosamine to GluA, all N-acetylglucosamine in peptidoglycan was included in GluA. DAPA was detected and quanti¢ed as £uorescent primary amines after derivatization with o-phthaldialdehyde [13,14]. A standard mixture containing LL, DD and meso DAPA eluted as two peaks, which were identi¢ed as meso and LL+DD DAPA. Dissolved combined amino acids (DCAA) in the rivers and the bacterial cultures (see below) were measured in 0.45-Wm ¢ltered samples that were hydrolyzed as above. The DCAA were detected and quanti¢ed by HPLC using the procedure for DAPA. 2.2. Bacterial cultures

2

Assuming (1) a bacterial density of 109 bacteria l31 , each with a C content of 10313 g, and a maximum of 2 mg dissolved organic carbon (DOC) l31 in pelagic waters, and (2) 109 bacteria g31 and a maximum of 5% organic C in marine sediments. Data compiled from various sources.

FEMSEC 1555 24-11-03

Bacterial strains used in the experimental work included Pseudomonas sp. (G3 ), Erythrobacter sp. (G3 ) and Bacillus sp. (Gþ ). The three strains were isolated from coastal or estuarine waters in Denmark and identi¢ed by 16S

Cyaan Magenta Geel Zwart

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

rDNA sequencing by L. Frette, National Environmental Research Institute, Roskilde, Denmark. 2.3. Degradation of whole bacterial cell walls Morphological changes of dead Bacillus and Erythrobacter cells due to degradation by natural estuarine bacteria were studied during a 2- and a half-month incubation period. The two bacterial strains were grown in 10% liquid Zobell medium [15] until stationary phase, washed three times in phosphate-bu¡ered saline (PBS ; 10 mM NaH2 PO4 in 0.9% NaCl at pH 7.5), and killed by exposure to UV light for 15 min (Philips 57413 P/30 30 W lamp). Reinoculation of subsamples of the UV-treated cells to 10% Zobell medium con¢rmed that all cells were killed by the light exposure. Killed cells were added to each of triplicate 500 ml 0.8-Wm ¢ltered water from Roskilde Fjord, Denmark, to a ¢nal density of 3.0U106 ml31 . The enrichment cultures were incubated at 20‡C on a horizontal shaker in the dark operating at 100 rpm. Samples for counting of all bacteria (estuarine bacteria and added dead bacteria [16]) and the added dead bacteria (immunochemical detection, see below) were collected every third day. Polyclonal antibodies against Bacillus sp. and Erythrobacter sp. were raised by immunizing rabbits with whole Bacillus cells as in [17] or with proteinase K-digested Erythrobacter cells as in [18]. Cells immobilized on 0.2-Wm pore-size polycarbonate ¢lters were detected by immuno£uorescence microscopy essentially as in [17] using the primary antibodies in dilutions of 1:1000. Fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulins (Dako, Glostrup, Denmark) diluted 1:20 were used as the secondary antibody. Bacterial cells on the ¢lters were recorded by a Leica TCS4d confocal laser scanning microscope at 1000U magni¢cation as in [19]. 2.4. Degradation of crude cell wall material Crude cell wall material of Pseudomonas sp., Erythrobacter sp. and Bacillus sp. was obtained from cultures grown in 10% liquid Zobell medium. The cells were harvested in the late exponential phase, washed twice in PBS and centrifuged at 8000Ug for 5 min. Finally the cells were sonicated (Sanyo Soniprep 150, 15 Wm amplitude) for six times 3 min in an ice bath, and subsequently autoclaved for 10 min to kill remaining living cells. The sonicated cell material was ¢ltered through 0.45-Wm ¢lters to remove larger cell fragments. In addition to cell wall material, the ¢ltrates probably also included various cytoplasmatic substances. Cell ¢ltrates corresponding to 6U109 Pseudomonas sp., 7.1U109 Erythrobacter sp. and 11.3U109 Bacillus sp. were each added to triplicate cultures of 250 ml Roskilde Fjord water (16 psu salinity). The water was ¢ltered through 0.8-Wm membrane ¢lters before the addition of cell ¢ltrate. As controls served triplicate

FEMSEC 1555 24-11-03

271

250 ml 0.8-Wm ¢ltered estuarine water without addition of cell ¢ltrate. All cultures were incubated for 8 days at 20‡C on a horizontal shaker in the dark at approximately 100 rpm. The 0.8-Wm ¢ltration of the Roskilde Fjord water was performed to remove bacterivorous protists. The proportion of estuarine bacteria removed by the ¢ltration was not measured, but when comparing the initial density of 1.2U106 ml31 in the 0.8-Wm ¢ltrate with the typical bacterial density in the fjord during the sampling period in June (6^9U106 ml31 ; unpublished), the ¢ltration may have removed about 85% of the ambient bacteria. Samples for counting estuarine bacteria were taken daily, and total bacterial counts were obtained by epi£uorescence microscopy after staining with acridine orange [16]. Water samples for analysis of peptidoglycan components were collected at days 0 and 8. The water samples were 0.45-Wm ¢ltered, hydrolyzed and analyzed by HPLC for content of D-AA, GluA and DAPA as above. At day 0 some estuarine bacteria may have been included in the 0.45-Wm ¢ltrates. At the end of the incubations, when signi¢cantly larger cells occurred in cultures, the ¢ltrates most likely did not include estuarine bacteria [20]. 2.5. Degradation of puri¢ed peptidoglycan Pseudomonas sp. and Bacillus sp. were grown in liquid Zobell medium and harvested in the late exponential phase. The cells were washed twice in PBS bu¡er and centrifuged at 8000Ug for 5 min before extraction of peptidoglycan according to Pelz et al. [21]. Brie£y, the extraction included (1) treatment with sodium dodecyl sulfate (SDS) at 100‡C for 1 h for cell lysis, (2) sonication for 1 min for further cell destruction, (3) digestion with trypsin to remove membrane proteins, and (4) extraction of lipids with organic solvents of decreasing polarity. The puri¢ed peptidoglycan was analyzed for content of AA, D-AA, GluA and DAPA by HPLC as above. Microbial degradation of the puri¢ed peptidoglycan was studied by addition of the peptidoglycan to 0.8-Wm ¢ltered estuarine water as described in Section 2.4. Triplicate cultures of 400 ml 0.8-Wm ¢ltered water received peptidoglycan corresponding to 1 mg l31 . All cultures were incubated for 116 days on a horizontal shaker at room temperature (20^25‡C) at 100 rpm in the dark. At day 105, 50 Wl L-broth (10 g tryptone l31 , 5 g yeast extract l31 , 10 g NaCl l31 and 1 g glucose l31 ) was added to the cultures to stimulate bacterial growth in an attempt to promote degradation of possible residual peptidoglycan in the cultures. Samples for bacterial counts and for analysis of amino compounds in the cultures were taken at variable time intervals. The collected water was analyzed for content of AA and GluA in un¢ltered samples (including dissolved and particulate matter) and in 0.45-Wm ¢ltered samples (dissolved fraction). Analysis of peptidoglycan components in un¢l-

Cyaan Magenta Geel Zwart

272

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

tered and ¢ltered water was performed since a portion of the puri¢ed peptidoglycan was found to consist of larger ( s 0.45 Wm) cell fragments. This was observed by epi£uorescence microscopy of acridine orange-stained peptidoglycan. The staining produced weakly £uorescent cell material that ranged from almost intact cell size to minute fragments. The particulate structure of a portion of the puri¢ed peptidoglycan implies that 0.45-Wm ¢ltered water from the estuarine cultures did not include all added peptidoglycan. Therefore both un¢ltered and 0.45-Wm ¢ltered water was analyzed to follow the degradation of peptidoglycan. 2.6. Comparison of peptidoglycan components in bacterial cell walls and in the Baltic rivers The composition of AA, D-AA, GluA and DAPA in puri¢ed peptidoglycan (Section 2.5) was related to the composition of peptidoglycan components in the Baltic rivers to estimate whether a selective degradation of peptidoglycan had occurred in the rivers.

3. Results and discussion 3.1. Peptidoglycan components in 50 Baltic rivers The concentration of D-AA and GluA in the Baltic rivers increased with the concentration of DON, while a different relationship emerged between DON and DAPA. D-AA ranged from 50 nM (most of the Swedish rivers) to 300 nM N (highest level in the Estonian and Latvian rivers) (Fig. 1A). Relative to the concentration of total DCAA in the rivers, the four D-AA on the average made up 5.5% (data not shown). The concentrations of GluA exceeded the amounts of D-AA and ranged from 500 (Swedish rivers) to about 2000 nM N (Finnish rivers) (Fig. 1B). For DAPA, one group of samples (13 rivers; half of the Finnish and the three Estonian rivers) had concentrations above 200 nM and appeared to vary independently of the DON concentrations (Fig. 1C). In the remaining rivers, DAPA concentrations were below 150 nM and correlated negatively with DON (r2 = 0.605).

Fig. 1. Concentrations of dissolved D-AA (sum of D-Asp, D-Glu, D-Ser and D-Ala; A), GluA (B), and DAPA (sum of meso and LL+DD isomers; C) relative to DON in 0.45-Wm ¢ltered water from 50 rivers discharging into the Baltic Sea. Numerals in the text box in A refer to the number of rivers sampled in each country. The linear regressions were calculated from all shown concentrations, except in C where DAPA concentrations s 150 nM (data points within the dotted line) were excluded. The samples were collected at the mouth of the rivers in July 1999. Details on sampling, water chemistry and concentrations of nutrients are given in Stepanaukas et al. [8]. Means T 1 S.D. shown. n = 3.

The exclusion of DAPA concentrations s 150 nM in the regression was chosen in an attempt to locate a relationship between DAPA and DON. The mechanisms causing the apparent lack of correlation between DAPA and DON (DAPA concentrations s 150 nM) or the inverse correlation (DAPA concentrations 6 150 nM) are unknown. Obviously the degradation or preservation of this peptidoglycan-speci¢c compound di¡ers from D-AA and GluA in natural waters. Possibly,

Table 1 Proportion of D-AA, DAPA and GluA at low and high DON concentrations in 50 Baltic rivers and in puri¢ed peptidoglycan of Pseudomonas sp. and Bacillus sp.

D-AA

DAPA GluA

Low DON (5 WM N)

High DON (55 WM N)

Relative composition of peptidoglycan in Pseudomonas sp. and Bacillus sp.

Relative composition of DON in the Baltic rivers vs. composition of peptidoglycan

% of DON

Ratio

% of DON

Ratio

Ratio

Ratio

0.37 T 0.06 1.0 T 0.35 0.28 T 0.03

1 2.7 7.6

0.51 T 0.21 0.08 T 0.06 10.8 T 4.10

1 0.22 29.2

1 0.083^0.066 0.178^0.406

1 32.5 (low DON)^3.33 (high DON) 42.7 (low DON)^71.9 (high DON)

Mean concentrations including 95% con¢dence interval ranges of D-AA, DAPA and GluA in the rivers were determined from the linear regressions in Fig. 1. Only DAPA concentrations 6 150 nM were included in the regression. Content of DAPA and GluA in the rivers and the bacteria was normalized to D-AA. The relative composition of peptidoglycan components was based on mean values of each bacterium in Table 2.

FEMSEC 1555 24-11-03

Cyaan Magenta Geel Zwart

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

dissolved DAPA, alone or associated with other peptidoglycan components, reacts with organic substances in some rivers and forms biologically refractory complexes. Although DAPA has been found preserved in ancient stromatolites [22], there are no reports of DAPA being recalcitrant to biological degradation and thus accumulate in nature. D-AA, GluA, and DAPA together comprised from 1.65 to 11.4% of DON in the rivers, indicating that bacterial cell wall components make up a sizable fraction of freshwater DON (regressions in Fig. 1, Table 1). This contribution of cell wall components is probably an underestimate of the share of peptidoglycan components in DON, since peptidoglycan also contains L-AA and other N compounds such as muramic acid. On the other hand, chitin (structural polymer in invertebrate exoskeletons and fungal cell walls [23]) may provide an additional source of GluA to the rivers. The present amounts of GluA were 10^30 times higher than concentrations published for high molecular mass (HMM) DOM (1^100 nm size) in the oceans [24]. However, relative to HMM DON, GluA in the oceanic samples made up about 0.5^5%, which agrees with the range measured in the Baltic rivers (0.28^10.8%; Table 1). The present DON pools were not separated into di¡erent molecular size classes, but assuming that most GluA resided in riverine HMM, e.g. in cell wall fragments, there is a close agreement between the riverine and the oceanic environments. The similarity may indicate that there was a minor terrestrial in£uence on the production of GluA in the rivers, e.g. due to a mainly bacterial origin of GluA. The abundance of GluA in the rivers may also support that GluA (and galactosamine) are important bacterial derived components of refractive DOM, as has been suggested for marine environment [3]. When normalized to D-AA, the content of DAPA and GluA was 3.33- to 32.5-fold and 42.7- to 71.9-fold higher, respectively, than in pure peptidoglycan of the bacteria Bacillus sp. and Pseudomonas sp. (Table 1). Thus, compared to the composition of AA in intact peptidoglycan (Tables 1 and 2), DON in the rivers was signi¢cantly en-

Table 2 Content of four D-AA, GluA and DAPA relative to the total D- and L-AA content in peptidoglycan of Bacillus sp. and Pseudomonas sp. % of all L+D-AA

D-Asp D-Glu D-Ser D-Ala

GluA DAPA

Bacillus sp.

Pseudomonas sp.

1.68 T 0.37 6.74 T 0.34 1.06 T 0.20 5.55 T 0.76 6.10 T 0.56 0.99 T 0.04

1.91 T 0.21 1.33 T 0.09 0.82 T 0.08 0.87 T 0.26 0.88 T 0.11 0.41 T 0.07

The total L- and D-AA concentrations were determined with the OPA derivatization procedure and do not include proline and cysteine. Mean T 1 S.D. shown. n = 3

FEMSEC 1555 24-11-03

273

riched with DAPA and GluA, relative to D-AA. In this comparison we did not consider a potential input of chitin-derived GluA, as well as selective degradation and abiotic sorption of peptidoglycan components may have occurred in the rivers. These processes could, together with biological degradation, have in£uenced the observed enrichment with GluA and DAPA in the rivers. Previous analyses of dissolved AA enantiomers in natural waters are few and indicate a variable occurrence. In marine waters, D-AA have been found either to be insigni¢cant (estuarine colloidal and particulate material [25]) or constitute a major portion of high molecular DON (oceanic environments [4]). No data on peptidoglycan components in freshwater are available, but the present study indicates that peptidoglycan components may account for a sizable portion of riverine DON. Peptidoglycan in the Baltic rivers may originate from aquatic as well as terrestrial environments, as D-AA have been shown to be released during £ooding of soil [26]. The biological availability of peptidoglycan components in the present rivers was not tested, but previous studies indicate that only a small fraction of riverine D-AA is degradable by microorganisms [26]. A recalcitrant nature of bacterial cell wall material is supported by the occurrence of nondegraded peptidoglycan components in the Baltic rivers. 3.2. Integrity of whole bacterial cell walls The riverine samples con¢rm that peptidoglycan and its constituents are not completely degraded by microorganisms, as has previously been observed in marine environments [4,7]. In order to determine to which extent bacterial cell walls and their peptidoglycan components are degradable by natural aquatic bacteria, the persistence of whole bacterial cell walls, cell wall material and puri¢ed peptidoglycan were studied in cultures of actively growing estuarine bacteria. The fate of whole, UV-killed cells of Erythrobacter sp. (G3 ) and Bacillus sp. (Gþ ), which were added to the estuarine cultures, was determined by immuno£uorescence microscopy. A progressive fragmentation of Bacillus sp. cell walls was observed during the 69-day period (Fig. 2, left panel). Cell fragments began to aggregate into amorphous structures after 3 days, and only clumps of cell debris remained present after 69 days. In contrast, the cell wall structure of Erythrobacter sp. remained intact during the entire incubation period (Fig. 2, right panel). During the experiment the total number of bacteria in the estuarine cultures (estuarine bacteria and stainable Erythrobacter sp. and Bacillus sp. cells) increased from about 4U106 cells ml31 at start to 15U106 cells ml31 (amendment with Bacillus sp.) and 9.6U106 cells ml31 (amendment with Erythrobacter sp.) at day 8. At day 69, cell densities of about 2U106 ml31 (Bacillus sp.) and 3.5U106 ml31 (Erythrobacter sp.) were found (data not shown).

Cyaan Magenta Geel Zwart

274

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

FEMSEC 1555 24-11-03

Cyaan Magenta Geel Zwart

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

The experiment showed that the estuarine bacteria rapidly degraded the cell walls of Bacillus sp., but did not degrade Erythrobacter sp. cell walls to a comparable extent. The employed polyclonal antibodies target several cell wall components. Therefore, the immuno£uorescence reaction monitors morphological changes rather than direct degradation of peptidoglycan during the incubation. However, peptidoglycan is responsible for the strength and shape of the Gþ cell wall [5]. Consequently the morphological changes of the Bacillus sp. cells shown in Fig. 2 most likely re£ect degradation or severe alteration of peptidoglycan. We speculate that lipopolysaccharides and proteins in the G3 Erythrobacter sp. outer membrane might protect its peptidoglycan against enzymatic attack, or that G3 peptidoglycan is not as readily degraded as Gþ peptidoglycan. The application of UV treatment to kill the bacteria was chosen as this procedure was expected to in£ict the least possible damage to the cell walls. Light-induced e¡ects on bacteria appear restricted to nucleic acids [27], but it cannot be excluded that the UV light had some e¡ects on degradability of the cell wall components. Peptidoglycan in Bacillus sp. spores has been found to resist extensive UV radiation but the peptidoglycan structure in spores and whole bacterial cells may not be similar [28]. In nature, morphologically intact but dead bacterial cells originate from mortality that only causes minor or no damage on the cell wall, e.g. starvation, predatory bacteria, antibiosis and possibly viral infection [29]. Bacterial ‘ghosts’ or nucleoid-less bacteria make up a substantial fraction (61^88%) of the total bacterial numbers in some marine waters [30]. Empty ‘cell sacs’ have also been observed in a marine sediment [31]. Our study suggests that cell walls of mainly G3 bacteria may retain their structural integrity for prolonged periods and thus contribute to the ‘ghost’ population in the environment. 3.3. Degradation of crude cell wall material The higher stability of G3 than of Gþ cells shown in Fig. 2 may indicate that G3 peptidoglycan better resisted microbial degradation than did Gþ peptidoglycan. To test this hypothesis, cultures of estuarine bacteria were amended with suspensions of ultrasonicated and 0.45-Wm ¢ltered cell wall material of Pseudomonas sp. and Erythrobacter sp. (G3 ) and Bacillus sp. (Gþ ). The amendments gave rise to a signi¢cant growth of estuarine bacteria. Hence, the initial density of 1.2U106 cells ml31 increased during the 8-day incubation to 54, 65 and 101U106 bacteria ml31 in the Pseudomonas sp., Erythrobacter sp. and Bacillus sp. amended cultures, respectively (Fig. 3A). In the controls the ¢nal density was 7.0U106 bacteria ml31 .

275

Fig. 3. Growth of estuarine bacteria and changes in concentrations of speci¢c peptidoglycan components after enrichment with crude cell wall material of Pseudomonas sp. (G3 ), Erythrobacter sp. (G3 ) and Bacillus sp. (Gþ ) during an 8-day period. Increase in bacterial density (A) and uptake of four D-AA (B), GluA (C) and meso and LL+DD DAPA (D) are shown in the panels. Means T 1 S.D. shown. n = 3 (one sample from each culture).

Simultaneous with the cell growth, the added peptidoglycan was reduced. After 8 days, 72% of D-AA and 89% of GluA were degraded in the cultures amended with Bacillus sp., while 51 and 45% (D-AA), and 69 and 65% (GluA) were degraded in the Pseudomonas sp. and Erythrobacter sp. cultures, respectively (Fig. 3B and C). Similarly, DAPA was reduced by 77% in the cultures amended with Bacillus sp., but only by 44 and 39% in the cultures with Pseudomonas sp. and Erythrobacter sp. (Fig. 3D). All AA concentrations were measured in the dissolved ( 6 0.45-Wm) fraction. The results indicate that peptidoglycan components derived from G3 cell walls were more resistant to microbial degradation than components of Gþ cell walls. No changes in D-AA, GluA or DAPA were found in the control cultures without enrichment with cell material.

6 Fig. 2. Confocal microscopy recordings of immunochemical slides showing morphological changes of dead Bacillus sp. (Gþ ) and Erythrobacter sp. (G3 ) during a 69-day incubation in cultures of estuarine bacteria.

FEMSEC 1555 24-11-03

Cyaan Magenta Geel Zwart

276

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

In addition to D-AA, GluA and DAPA, the cell wall material also included DCAA, originating from bacterial proteins and the estuarine water. The initial DCAA concentrations were 7-fold higher (mean value) than the D-AA concentrations (Table 3). At day 8, the DCAA concentrations were only 2.8-fold higher (mean value) than the D-AA, indicating that DCAA were taken up to a larger extent than D-AA by the bacteria. In the controls, the DCAA were reduced by 40%. The concentration of DON in the estuarine water was 37 WM N (data not shown). Changes in DON due to addition of peptidoglycan and during the experiments were not followed. The amount of Bacillus sp. cell wall material added to the cultures was 2.5- to 3-fold higher than that of the other two bacteria (exempli¢ed by the concentrations of D-AA, GluA and DAPA at day 0). This di¡erence in cell wall material may have in£uenced the rate of degradation, but it appeared not to a¡ect the overall degradation of D-AA, GluA and DAPA, as comparable, residual concentrations of these amino compounds were present in the three cultures at day 8. Thus, for testing degradability of G3 and Gþ cell wall components, the di¡erent amounts of cell wall material seemed not to introduce any experimental biases. Interestingly, ultrasonication released about 80% of the total DAPA content in Bacillus sp. into the 6 0.45-Wm fraction, while less than 30% of DAPA in Pseudomonas sp. and Erythrobacter sp. was released. This demonstrates that DAPA is more ¢rmly integrated into the G3 lipopolysaccharide^protein cell wall matrix, than in the peptidoglycan-dominated Gþ cell wall. The relative high abundance of DAPA in Bacillus sp. (made up about 1% of all peptidoglycan AA ; Table 2) was unexpected as this AA according to literature is a typical component of G3 peptidoglycan and is replaced by lysine in Gþ bacteria [5]. However, DAPA does occur in peptidoglycan of some Gþ bacteria [32] and is used as a chemotaxonomic marker in Actinobacteria (Gþ bacteria ; [33]). The occurrence of DAPA in Gþ bacteria in natural environments is unknown. However, the low degradation of G3 peptidoglycan observed in this study and the dominance of G3 bacteria in aquatic environments [34] both support that the abundant amounts of DAPA in the Baltic rivers most likely originated from G3 bacteria. Table 3 Initial and ¢nal concentrations of and 25a )

D-AA

3.4. Degradation of puri¢ed peptidoglycan The previously shown modi¢cations of whole bacterial cell walls and the degradation of crude cell wall material suggest that Gþ peptidoglycan is less resistant to microbial degradation than that of G3 bacteria. To test if pure peptidoglycan of Gþ bacteria actually is more labile than that of G3 bacteria, puri¢ed peptidoglycan of Pseudomonas sp. and Bacillus sp. was added to cultures of estuarine bacteria. This experiment was followed for 116 days. Results from the nutrient addition on day 105 are given after presentation of results from the initial 105-day period. 3.4.1. Changes in bacterial growth and peptidoglycan components between days 0 and 105 The addition of peptidoglycan signi¢cantly stimulated growth of the estuarine bacteria. After 6 days, the bacterial abundance had increased from initially 0.15 to 2.4U106 cells ml31 (controls without peptidoglycan) and 0.85 to 8.3 and 9.9U106 cells ml31 in the cultures with peptidoglycan of Pseudomonas sp. and Bacillus sp., respectively (Fig. 4A). For unknown reasons, the initial bacterial density was lower in the controls than in the peptidoglycan-enriched cultures. However, the about 4-fold higher bacterial abundance in the enriched cultures than in the controls at day 6 demonstrates that peptidoglycan sustained a larger bacterial growth. At 105 days, the bacterial abundance in the peptidoglycan-enriched cultures had decreased to 1.1^1.2U106 cells ml31 and 0.3U106 cells ml31 in the controls. The initial amounts of D-AA in the un¢ltered water (about 1000 nM; Fig. 4B) and 0.45-Wm ¢ltered water (75^150 nM; Fig. 4C) demonstrate that the majority of peptidoglycan-derived AA resided in the particulate fraction ( s 0.45 Wm). The un¢ltered water includes the puri¢ed peptidoglycan, naturally occurring peptidoglycan in the fjord water, and peptidoglycan in the estuarine bacteria; the 0.45-Wm ¢ltered water includes the corresponding, dissolved peptidoglycan pools (expected to be free of bacteria) in the cultures. During the following 4 days, the amount of D-AA in the un¢ltered water declined, reducing the concentration by 510 nM (Bacillus sp.) and 390 nM (Pseudomonas sp.). Concentration changes in the controls were not statistically signi¢cant (P s 0.05; t-test).

and DCAA during degradation of crude cell wall material (days 0 and 8) and puri¢ed peptidoglycan (days 0

Degradation of crude cell wall material (Fig. 3)

Day 0

D-AA

Day 8 or 25

DCAA D-AA DCAA

Degradation of pure peptidoglycan (Fig. 4)

Pseudomonas sp.

Erythrobacter sp.

Bacillus sp.

Control

Pseudomonas sp.

Bacillus sp.

Control

889 T 57 5 994 T 741 392 T 68 1 447 T 187

1 245 T 231 7 618 T 521 611 T 129 1 390 T 215

2 950 T 352 21 063 T 1921 845 T 120 2 274 T 321

219 T 17 1 633 T 102 258 T 48 981 T 157

86 T 21 1 147 T 82 168 T 31 1 529 T 193

140 T 21 1 459 T 153 142 T 23 1 568 T 179

65 T 27 1 063 T 118 112 T 32 1 352 T 83

All concentrations are given in nM concentrations of 0.45-Wm ¢ltered samples. S.D. shown; n = 3. a End of the initial sampling period.

FEMSEC 1555 24-11-03

Cyaan Magenta Geel Zwart

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

277

The reduction of the added peptidoglycan re£ects bacterial uptake of D-AA, but the actual amounts taken up cannot be determined as the concentration changes include both reduction of the added and naturally occurring peptidoglycan, and the increase in peptidoglycan in the growing estuarine bacteria. The reduction in D-AA concentrations corresponded to a decline of 39% (Pseudomonas sp.) and 49% (Bacillus sp.) of the initial peptidoglycan D-AA. Between days 4 and 25, D-AA in the un¢ltered samples either increased due to the ongoing bacterial production (Bacillus sp.) or remained largely unchanged (Pseudomonas sp. and controls). In the 0.45-Wm ¢ltered water, an initial reduction of D-AA of 140^90 nM was observed for Bacillus sp. peptidoglycan, but otherwise D-AA were released and increased the concentrations from initially about 90 to 170 nM on day 25 (Fig. 4C). On days 9, 16 and 25, concentrations of D-AA were 16^27% higher in the cultures with Pseudomonas sp. than with Bacillus sp. peptidoglycan. The addition of peptidoglycan to the cultures also included DCAA, but a higher DCAA concentration was only found in the Bacillus sp. amendment (27% more DCAA at day 0) (Table 3). During the 25-day incubation period, the DCAA concentration slightly increased (Pseudomonas sp. cultures and control) or remained unchanged (Bacillus sp. cultures). The results indicate that there was no net uptake of DCAA in the estuarine water. The DON concentration in the estuarine water was 49 WM N, but changes in concentrations were not measured in the experiment. Presence of another peptidoglycan component, GluA, in the un¢ltered samples increased 2.2-fold (controls) and up to 7.6-fold (Bacillus sp.) during the initial 25 days, followed by a decline until day 105 (Fig. 4D). The initial amounts of GluA were rather similar in the controls (230 nM) and in the cultures enriched with peptidoglycan (250^350 nM), suggesting that the added peptidoglycan had a low content of GluA. Changes in concentrations of dissolved GluA in the 6 0.45-Wm ¢ltered samples showed that a production as well as an uptake of GluA occurred. During the initial 16 days, the amount of dissolved GluA increased 2.0- to 3.75-fold (concentration range of 120^550 nM), but was followed by a decrease (Fig. 4E).

Fig. 4. Growth of estuarine bacteria and changes in concentrations of four D-AA (D-Asp, D-Glu, D-Ser and D-Ala) and GluA in un¢ltered and 0.45-Wm ¢ltered water after enrichment with puri¢ed peptidoglycan of Pseudomonas sp. (G3 ) and Bacillus sp. (Gþ ) (equivalent to 1 mg l31 ) during a 116-day period. The cultures were enriched with L-broth at day 105. Increase in bacterial density (A), D-AA in un¢ltered (B) and 0.45-Wm ¢ltered water (C), and GluA in un¢ltered (D) and 0.45-Wm ¢ltered water (E) are shown in the panels. Means T 1 S.D. shown. n = 3 (one sample from each culture).

FEMSEC 1555 24-11-03

3.4.2. Changes in bacterial growth and peptidoglycan components between days 105 and 116 The addition of growth medium (L-broth) on day 105 caused a considerable regrowth of the estuarine bacteria. The cell density increased from about 106 cells ml31 (or below) to 0.7^2.5U108 cells ml31 on day 116 (Fig. 4A). Simultaneous with the bacterial growth, the concentrations of D-AA in the un¢ltered water increased from about 500 nM to levels of 3000^5000 nM on day 107 (Fig. 4B). These D-AA were reduced by 43^51% on day 112 in the Bacillus sp. and control cultures ; no changes occurred in the Pseudomonas sp. cultures. Similarly, in the 6 0.45-Wm

Cyaan Magenta Geel Zwart

278

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

¢ltered water, the D-AA content increased from 50^100 nM at day 105, to 3500^5000 nM at day 107 (Fig. 4C). However, these D-AA were signi¢cantly reduced the following days, concurrent with the increase in bacterial density. The nutrient enrichment also led to a signi¢cant raise in GluA. In the un¢ltered water, the concentration of GluA increased 20- to 42-fold after day 105 (Fig. 4D). In contrast to D-AA in the un¢ltered water, there were no indications of a bacterial utilization of ‘un¢ltered’ GluA in the cultures. In the 6 0.45-Wm fraction, the concentration of GluA increased to about 15 000 nM (Fig. 4E). Interestingly, largely all (77^90%) of this GluA was assimilated by the growing bacteria during the following 9 days. Assuming a bacterial carbon content of 10313 g cell31 and a carbon content of GluA of 72 ng C nM31 , the uptake of dissolved GluA corresponded to approximately 10% of the net bacterial C production. 3.4.3. Degradation and production of peptidoglycan components in the 116-day experiment The main conclusions from the enrichment with peptidoglycan are (1) that about half of the D-AA in peptidoglycan were degradable, (2) that D-AA of peptidoglycan from the Gþ Bacillus sp. were assimilated to a larger extent than D-AA of the G3 Pseudomonas sp., and (3) that more D-AA of Pseudomonas sp. than of Bacillus sp. accumulated in the 6 0.45-Wm fraction during the initial 25-day period. These observations support that Gþ cell walls, including their peptidoglycan, are more labile than cell walls of G3 bacteria. The almost identical amounts of added peptidoglycan of the two bacteria were re£ected in similar bacterial abundances within the initial 25 days. The initial concentrations of dissolved D-AA and GluA were more variable, but obviously they did not in£uence the bacterial production within the same period. Our experiments do not reveal whether peptidoglycan can be totally degraded by bacterial processes, as the added peptidoglycan could not be size-separated from peptidoglycan in living bacteria. However, the residual concentrations of dissolved D-AA (about 100 nM) and GluA (about 200 nM) after 105 days of incubation were close to the ambient concentrations before the addition of peptidoglycan. This suggests that most of the peptidoglycan in the dissolved fraction, being added or produced during the incubation (Fig. 4C and E), was degraded. The residual amounts of D-AA and GluA after 105 days show that portions or constituents of peptidoglycan are resistant to bacterial degradation. Possibly, due to their polymer structure, these compounds are not easily degraded by bacterial enzymes, such as hydrolases, as has been suggested to explain the presence of peptidoglycan in natural environments [35]. The semilabile properties of peptidoglycan observed in our study are supported by long turnover times (10^167 days) recently observed for radiolabelled G3 peptidoglycan in marine waters [36].

FEMSEC 1555 24-11-03

The study further demonstrated a slower degradation of the polysaccharide than the peptide moiety of G3 peptidoglycan. If the similar is true of freshwater environments, a more recalcitrant nature of saccharide components in peptidoglycan may explain the higher abundance of GluA than of D-AA in the rivers (Fig. 1). The release of dissolved D-AA and GluA following the regrowth of bacteria after enrichment with L-broth shows that fast-growing bacteria may release cell wall material, the process being deliberate or accidental. The subsequent re-assimilation of D-AA and GluA con¢rms that bacteria can take up extracellular peptidoglycan components. These D-AA and GluA most likely resided in intact or fragments of peptidoglycan, as a chromatographic analysis demonstrated that they did not occur as free monomers (data not shown). Once degraded to free molecules, e.g. by bacterial ectoenzymes, both D-AA and GluA are taken up by bacterial populations [37^39]. An additional process that might have in£uenced the AA pools in the cultures is virus-induced lysis of the bacteria. Weinbauer and Peduzzi [40] observed an increase in dissolved AA following addition of virus-rich material to natural marine cultures. If lysogenic cells, or viruses and their bacterial hosts, were present in the peptidoglycanenriched cultures, production of cell fragments from bacterial lysis, also including peptidoglycan components, may have contributed to the observed increase of both dissolved D-AA and GluA during the initial 25-day period. The importance of viral infection on the degradation of peptidoglycan in natural waters has not been studied, but its signi¢cance may be underestimated and deserves further analysis. 3.5. Enzymatic reactions and peptidoglycan degradation In contrast to the addition of whole bacterial cells and crude peptidoglycan to cultures of estuarine bacteria (Figs. 2 and 3), the addition of puri¢ed peptidoglycan did not include additional proteins (as structural constituents or enzymes), lipids or other cell components. The signi¢cance of these non-peptidoglycan products on the degradation of peptidoglycan in whole cells and crude cell material cannot be estimated from the present experiments. Possibly, non-peptidoglycan compounds may have served as nutrients and stimulated the bacterial growth and with this the activity of enzymes involved in degradation of peptidoglycan. Enzymatic processes involved in uptake of extracellular peptidoglycan are still unidenti¢ed. Among bacterial enzymes known to be active in peptidoglycan degradation are hydrolases that break covalent bonds in peptidoglycan. These enzymes are typically located in the cell wall [41,42]. Another process for peptidoglycan hydrolysis is found in bacteriophages that produce lytic enzymes which create small openings in peptidoglycan [43]. If similar cellbound enzyme systems are responsible for degradation of

Cyaan Magenta Geel Zwart

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

extracellular peptidoglycan, a physical contact between the bacterial cells and peptidoglycan is necessary, but the mechanisms are unknown. An alternate pathway for degradation of peptidoglycan may involve a suite of extracellular enzymes such as peptidases (for removal of the peptide interbridges) and lysozyme-like enzymes (for breakage of the L(1-4) bonds between N-acetylglucosamine and N-acetylmuramic acid). Both peptidases and L(1-4)-cleaving enzymes are commonly found in natural waters [44,45]. Another aspect that may in£uence the enzymatic attack on peptidoglycan is composition of the extracted peptidoglycan. In our study, the cell wall material was digested with trypsin to remove membrane proteins [21]. Other studies, e.g. Nagata et al. [36], did not include an enzymatic treatment of the cell wall material. We did not test to which extent trypsin actually removed membrane proteins or perhaps even modi¢ed peptidoglycan. If a physical contact between extracellular peptidoglycan and cellbound peptidoglycan hydrolases is required, presence of membrane proteins may in£uence the enzymatic activity. The present lack of standard procedures for extraction of peptidoglycan restricts a comparison of results on degradation of peptidoglycan and deserves more attention.

279

degradation. Results from the experiments further indicate that cell walls of G3 bacteria are more resistant to degradation than those of Gþ bacteria. This may be related to the di¡erences in peptidoglycan structure between G3 and Gþ bacteria. The present knowledge on occurrence and cycling of peptidoglycan in natural environments is very restricted and requires additional research. Our observations on peptidoglycan components in the Baltic rivers and the degradation of cell wall material demonstrate that although peptidoglycan-derived components are not dominant elements of DOM, they may be important in shaping the composition and reactivity of natural DOM.

Acknowledgements We thank R.E. Jensen for technical assistance, L. Frette for donating bacterial cultures, and professors D. Burdige, C. Klausen and M.A. Moran for valuable comments on the manuscript. The study was supported by Danish Natural Science Research Council, MISTRA, the foundation Oscar och Lili Lamms Minne, the EU TMR research network ‘Wetland Ecology and Technology’ and the Swedish Institute.

3.6. Pre¢ltering and bacterial grazing In an attempt to eliminate protist grazing in the degradation experiments, estuarine bacteria in 0.8-Wm ¢ltered rather than natural, un¢ltered water was used in the cultures. When present in bacterial laboratory cultures, bacterivorous protists may signi¢cantly reduce bacterial populations [46]. In the present experiments, utilization of cell wall material by the estuarine bacteria was expected to result in increased bacterial densities. However, with grazing protists present, the bacterial density might not mirror the actual bacterial production. The 0.8-Wm pre¢ltration was estimated to exclude about 85% of the bacteria, including mainly attached and large bacteria. This means that bacteria in the cultures represent only a minor portion of the natural populations and that the observed degradation of cell wall material may not be directly comparable to the degradation at in situ conditions. The ¢ltration may introduce a major bias if attached and large bacteria are dominant organisms in the degradation of cell wall material. This was not tested in the present experiments. 3.7. Conclusions and perspectives The occurrence of peptidoglycan components in the Baltic rivers, the ocean [4], and marine sediments [7,47] corroborates that peptidoglycan is not completely degradable by microorganisms and contributes to the pool of recalcitrant organic matter. Our studies on degradation of peptidoglycan support that a fraction of peptidoglycan, exempli¢ed by D-AA and GluA, is resistant to bacterial

FEMSEC 1555 24-11-03

References [1] Tanoue, E. (1995) Detection of dissolved protein molecules in oceanic waters. Mar. Chem. 51, 239^252. [2] Nagata, T. and Kirchman, D.L. (2001) Bacterial mortality : A pathway for the formation of refractory DOM ? In: Microbial Biosystems: New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology Vol. 1, pp. 153^158. [3] Ogawa, H., Amagai, Y., Koike, I., Kaiser, K. and Benner, R. (2001) Production of refractory dissolved organic matter by bacteria. Science 292, 917^920. [4] McCarthy, M.D., Hedges, J.I. and Benner, R. (1998) Major bacterial contribution to marine dissolved organic nitrogen. Science 281, 231^ 234. [5] Madigan, M.T., Martinko, J.M. and Parker, J. (2000) BROCK Biology of Microorganisms, 9th Edn., pp. 1^991. Prentice-Hall, Upper Saddle River, NJ. [6] Lee, C. and Bada, J.L. (1977) Dissolved amino acids in the equatorial Paci¢c, the Sargasso Sea, and Biscayne Bay. Limnol. Oceanogr. 22, 502^510. [7] Pedersen, A.G.U., Thomsen, T.R., Lomstein, B.Aa. and Jrgensen, N.O.G. (2001) Bacterial in£uence on amino acid enantiomerization in a coastal marine sediment. Limnol. Oceanogr. 46, 1358^1369. [8] Stepanauskas, R., Jrgensen, N.O.G., Eigaard, O.R., Zvikas, A., Tranvik, L.J. and Leonardson, L. (2002) Summer inputs of riverine nutrients to the Baltic Sea: Bioavailability and eutrophication relevance. Ecol. Monogr. 72, 579^597. [9] Burdige, D.J. and Zheng, S.L. (1998) The biogeochemical cycling of dissolved organic nitrogen in estuarine sediments. Limnol. Oceanogr. 43, 1796^1813. [10] Jrgensen, N.O.G. and Jensen, R.E. (1997) Determination of dissolved combined amino acids using microwave-assisted hydrolysis and HPLC precolumn derivatization for labeling of primary and secondary amines. Mar. Chem. 57, 287^297.

Cyaan Magenta Geel Zwart

280

N.O.G. J8rgensen et al. / FEMS Microbiology Ecology 46 (2003) 269^280

[11] Bru«ckner, H., Haasmann, S., Langer, M., Westhauser, T., Wittner, R. and Godel, H. (1994) Liquid-chromatographic determination of D-amino and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J. Chromatogr. A 666, 259^273. [12] Mopper, K. and Furton, K.G. (1999) Extraction and analysis of polysaccharides, chiral amino acids, and SFE-extractable lipids from marine POM. Geophys. Monogr. 63, 151^160. [13] Lindroth, P. and Mopper, K. (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn £uorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667^1674. [14] Jrgensen, N.O.G., Kroer, N., Co⁄n, R.B., Yang, X.H. and Lee, C. (1993) Dissolved free amino acids, combined amino acids, and DNA as sources of carbon and nitrogen to marine bacteria. Mar. Ecol. Prog. Ser. 98, 135^148. [15] ZoBell, C. (1946) Marine Microbiology; a Monograph on Hydrobacteriology. Chronica Botanica Co., Waltham, MA. [16] Hobbie, J.E., Daley, R.J. and Jasper, S. (1977) Use of nucleopore ¢lters for counting bacteria by £uorescence microscopy. Appl. Environ. Microbiol. 33, 1225^1228. [17] Nybroe, O., Christo¡ersen, K. and Riemann, B. (1992) Survival of Bacillus licheniformis in seawater model ecosystems. Appl. Environ. Microbiol. 58, 252^259. [18] Middelboe, M., Jrgensen, N.O.G. and Kroer, N. (1996) E¡ects of viruses on nutrient turnover and growth e⁄ciency of noninfected marine bacterioplankton. Appl. Environ. Microbiol. 62, 1991^1997. [19] Hansen, M., Kragelund, L., Nybroe, O. and Srensen, J. (1997) Early colonization of barley roots by Pseudomonas £uorescens studied by immuno£uorescence technique and confocal laser scanning microscopy. FEMS Microbiol. Ecol. 23, 353^360. [20] Kroer, N. (1993) Bacterial growth e⁄ciency on natural dissolved organic matter. Limnol. Oceanogr. 38, 1282^1290. [21] Pelz, O., Cifuentes, L.A., Hammer, B.T., Kelley, C.A. and Co⁄n, R.B. (1998) Tracing the assimilation of organic compounds using delta C-13 analysis of unique amino acids in the bacterial peptidoglycan cell wall. FEMS Microbiol. Ecol. 25, 229^240. [22] Borruat, G., Roten, C.A.H., Marchant, R., Fay, L.B. and Karamata, D. (2001) Chromatographic method for diaminopimelic acid detection in calcareous rocks ^ Presence of a bacterial biomarker in stromatolites. J. Chromatogr. A 922, 219^224. [23] Horton, H.R., Moran, L.A., Ochs, R.S., Rawn, J.D. and Scrimgeour, K.G. (2002) Principles of Biochemistry, 3rd Edn., pp. 1^862. Prentice-Hall, Upper Saddle River, NJ. [24] Benner, R. and Kaiser, K. (2003) Abundance of amino sugars and peptidoglycan in marine particulate and dissolved organic matter. Limnol. Oceanogr. 48, 118^128. [25] Sigleo, A.C., Hare, P.E. and Helz, G.R. (1983) The amino acid composition of estuarine colloidal material. Estuar. Coast. Shelf Sci. 17, 87^96. [26] Stepanauskas, R., Laudon, H. and Jrgensen, N.O.G. (2000) High DON bioavailability in boreal streams during a spring £ood. Limnol. Oceanogr. 45, 1298^1307. [27] Je¡rey, W.H., Pledger, R.J., Aas, P., Hager, S., Co⁄n, R.B., Von Haven, R. and Mitchell, D.L. (1996) Diel and depth pro¢les of DNA photodamage in bacterioplankton exposed to ambient solar ultraviolet radiation. Mar. Ecol. Prog. Ser. 137, 283^291. [28] Atrih, A. and Foster, S.J. (2002) Bacterial endospores the ultimate survivors. Int. Dairy J. 12, 217^223. [29] Fuhrman, J.A. and Noble, R.T. (2001) Causative agents of bacterial mortality and the consequences to amine food webs. In: Microbial

FEMSEC 1555 24-11-03

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43] [44] [45]

[46]

[47]

Biosystems : New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology Vol. 1, pp. 145^151. [ ., Pinhassi, J. and Zweifel, U.L. (2001) Marine bacteHagstro«m, A rioplankton show bursts of rapid growth induced by substrate shifts. Aquat. Microb. Ecol. 24, 109^115. Moriarty, D.J.W. and Hayward, A.C. (1982) Ultrastructure of bacteria and the proportion of gram-negative bacteria in marine sediments. Microb. Ecol. 8, 1^14. McKerrow, J., Vagg, S., McKinney, T., Seviour, E.M., Maszenan, A.M., Brooks, P. and Seviour, R.J. (2000) A simple HPLC method for analysing diaminopimelic acid diastereomers in cell walls of Gram-positive bacteria. Lett. Appl. Microbiol. 30, 178^182. Goodfellow, M. and LeChevalier, M.P. (1986) Genus Nocardia Trevisan 1889. In: Bergey’s Manual of Systematic Bacteriology (Sneath, P.H.A., Ed.), pp. 1459^1471. WilliamspWilkins, Baltimore, MD. Hagstrom, A., Pinhassi, J. and Zweifel, U.L. (2000) Biogeographical diversity among marine bacterioplankton. Aquat. Microb. Ecol. 21, 231^244. de Leeuw, J.W. and Largeau, C. (1993) A review of macromolecular organic compounds that comprise living organisms and their role in kerogen, coal, and petroleum formation. In: Organic Geochemistry (Engel, M.H. and Macko, S.A., Eds.), pp. 23^72. Plenum Press, New York. Nagata, T., Meon, B. and Kirchman, D.L. (2003) Microbial degradation of peptidoglycan in seawater. Limnol. Oceanogr. 48, 745^ 754. Bunte, C. and Simon, M. (1999) Bacterioplankton turnover of dissolved free monosaccharides in a mesotrophic lake. Limnol. Oceanogr. 44, 1862^1870. O’Dowd, R.W. and Hopkins, D.W. (1998) Mineralization of carbon from D- and L-amino acids and D- glucose in two contrasting soils. Soil Biol. Biochem. 30, 2009^2016. Riemann, L. and Azam, F. (2002) Widespread N-acetyl-D-glucosamine uptake among pelagic marine bacteria and its ecological implications. Appl. Environ. Microbiol. 68, 5554^5562. Weinbauer, M.G. and Peduzzi, P. (1995) E¡ect of virus-rich high molecular weight concentrates of seawater on the dynamics of dissolved amino acids and carbohydrates. Mar. Ecol. Prog. Ser. 127, 245^253. Cibik, R., Tailliez, P., Langella, P. and Chapot-Chartier, M.P. (2001) Identi¢cation of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum. Appl. Environ. Microbiol. 67, 858^864. Smith, T.J., Blackman, S.A. and Foster, S.J. (2000) Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146, 249^262. Rydman, P.S. and Bamford, D.H. (2002) Phage enzymes digest peptidoglycan to deliver DNA. ASM News 68, 330^335. Karner, M., Fuks, D. and Herndl, G.J. (1992) Bacterial activity along a trophic gradient. Microb. Ecol. 24, 243^257. Zubkov, M.V. and Sleigh, M.A. (1998) Heterotrophic nanoplankton biomass measured by a glucosaminidase assay. FEMS Microbiol. Ecol. 25, 97^106. Hahn, M.W., Moore, E.R.B. and Ho«£e, M.G. (2000) Role of microcolony formation in the protistan grazing defense of the aquatic bacterium Pseudomonas sp. MWH1. Microb. Ecol. 39, 175^185. Grutters, M., van Raaphorst, W., Epping, E., Helder, W., de Leeuw, J.W., Glavin, D.P. and Bada, J.L. (2002) Preservation of amino acids from in situ-produced bacterial cell wall peptidoglycans in northeastern Atlantic continental margin sediments. Limnol. Oceanogr. 47, 1521^1524.

Cyaan Magenta Geel Zwart