National Institute of Oceanography, Dona Paula, Goa 403 004, India.
Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay1
Article first published online: 5 JAN 2006
FEMS Microbiology Ecology
Volume 34, Issue 1, pages 63–71, October 2000
How to Cite
Ramaiah, N., Hill, R. T., Chun, J., Ravel, J., Matte, M. H., Straube, W. L. and Colwell, R. R. (2000), Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay. FEMS Microbiology Ecology, 34: 63–71. doi: 10.1111/j.1574-6941.2000.tb00755.x
Contribution No. 275 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute.
- Issue published online: 5 JAN 2006
- Article first published online: 5 JAN 2006
- Received 8 July 2000, Revised 6 August 2000, Accepted 10 August 2000
- chiA gene;
- Chesapeake Bay
PCR primers specific for the chiA gene were designed by alignment and selection of highly conserved regions of chiA sequences from Serratia marcescens, Alteromonas sp., Bacillus circulans and Aeromonas caviae. These primers were used to amplify a 225 bp fragment of the chiA gene from Vibrio harveyi to produce a chiA gene probe. The chiA PCR primers and probe were used to detect the presence of the chiA gene in an assemblage of 53 reference strains and gave consistent results. Selected chiA fragments amplified by PCR were cloned and sequenced from nine known strains and from Chesapeake Bay isolates 6d and 11d. This confirmed the specificity and utility of the primers for detection of chiA-positive environmental strains. Over 1000 bacterial isolates from Chesapeake Bay water samples were tested for the presence of the chiA gene which was found to be present in 5–41% (average 21%) of the culturable bacterial community. The approach developed in this study was valuable for isolation and enumeration of chiA-positive bacteria in environmental samples.
Free-living marine and estuarine bacteria are pivotal in the biogeochemical cycling of nutrients [1,2]. Breakdown and biotransformation of marine autochthonous, complex organic molecules, such as chitin, alginate, and pectin, are mediated primarily by bacterial communities via catabolic enzyme systems. In the marine environment, chitin is an abundant complex carbohydrate polymer, being the principal exoskeletal material of crustaceans and many mollusks. The dry weight of chitin produced as a result of molting by the planktonic crustacean, Euphausia pacifica, alone has been estimated at 5–12 million tons annually [3,4]. Thus, conversion of chitin into utilizable forms by marine chitinolytic bacteria is essential in the recycling of carbon, nitrogen, and energy in the marine environment .
Some ecological studies have investigated the prevalence of chitin-degrading bacteria and rates of chitin degradation in the environment [6–10]. However, there have been few attempts to apply molecular approaches to ecological studies of chitinolytic bacteria in the marine environment, using, for example, nucleic acid probes for detection of bacteria possessing genes encoding for chitinase. Detection and quantitative determination of the presence (or absence) of a gene encoding a particular characteristic can be achieved with great speed and accuracy by nucleic acid hybridization, thus circumventing many limitations associated with conventional bacteriological methods [11,12].
Chitin is a complex polymer consisting of N-acetylglucosamine chains arranged in an antiparallel (α) or a parallel (β) configuration with differing degrees of deacetylation. The presence of associated proteins further adds to its complexity. Bacteria generally possess several chitinase genes encoding enzymes with diverse chitinase activities. Chitinase genes from several marine bacteria have been characterized [13–17]. The complete hydrolysis of chitin requires two enzymes, chitinase and hexosaminidase. Hexosaminidases all have a common evolutionary origin and no relationship to chitinases . In the case of the marine bacterium Alteromonas sp. strain O-7, at least four different chitinases (ChiA, ChiB, ChiC, and ChiD) and three different N-acetylglucosaminidases (GluNAcaseA, GluNAcaseB, and GluNAcaseC) are produced in the presence of chitin . Vibrio harveyi produces about 10 chitinases when grown on chitin, six of which have been cloned and identified. The sequence of chitinase in Vibrio cholerae has been identified in the complete genome sequence of this bacterium (Heidleberg, Clayton, Colwell, and Frasier, work in progress). Separate chitinases in V. harveyi and probably other marine bacteria are likely to be synthesized for efficient utilization of different forms of chitin .
We selected the chiA gene as our target gene since the availability of chiA sequences from several distantly related bacteria facilitated selection of highly conserved regions of the chiA gene for design of PCR primers expected to have broad specificity for detection of chiA in a wide range of bacteria. Our aims were to construct a chiA gene probe for the reliable detection of this gene in environmental bacterial strains and to use this gene probe for detecting and enumerating chiA genotypes in the culturable heterotrophic bacterial community from the Chesapeake Bay.
2Materials and methods
2.1Test strains and growth conditions
Bacterial strains used as reference strains are listed in Table 1. Strains were grown in Luria-Bertani (LB) broth  overnight with shaking at 30°C for DNA extractions and were maintained on LB agar plates.
|Species and strain number||chiAa||Chitin substrateb|
|A. hydrophila ATCC1546||−||−||+||+|
|A. hydrophila ATCC7966||+||+||+||+|
|Beneckea campbellii ATCC25920||+||+||+||−|
|Escherichia coli ATCC34309||−||−||−||−|
|E. coli O157||−||−||+||−|
|Klebsiella pneumoniae ATCC13883T||+||+||−||+|
|P. shigelloides UM6451||+||+||−||−|
|Pseudomonas aeruginosa ATCC27852||−||−||−||−|
|Vibrio aestuarianus ATCC3504||+||+||−||−|
|Vibrio alginolyticus ATCC17749||+||+||−||−|
|Vibrio anguillarum ATCC19105||−||−||−||−|
|V. anguillarum UM2445||+||+||+||+|
|V. cholerae UM400||−||−||−||−|
|V. cholerae UM928||−||−||−||+|
|V. cholerae O139 UMNT330||−||−||−||−|
|V. cholerae O139 Al1877||+||+||+||−|
|V. cholerae O1 088||−||−||+||−|
|V. cholerae O1 0100||+||+||+||−|
|V. cholerae O1 6367||−||−||+||−|
|V. cholerae O1 ATCC11623||−||−||+||−|
|V. cholerae O1 El Tor ATCC39315||+||+||−||−|
|V. cholerae non O1 ATCC14547||−||−||+||−|
|V. cholerae non O1 ATCC25872||−||−||−||+|
|V. cholerae ATCC14035T||+||+||+||−|
|V. cholerae ATCC25574||+||+||−||+|
|V. cholerae O31Y1 NRT 36S||−||−||+||+|
|V. cholerae IGR TMA21||−||−||−||−|
|V. cholerae Y334||+||+||−||−|
|Vibrio damselae UM4236||+||+||+||+|
|Vibrio diazotrophicus UM4232||−||−||−||−|
|V. fischeri UM1373||+||+||−||−|
|Vibrio furnissii UM4367||−||−||−||+|
|V. harveyi ATCC14216||+||+||+||−|
|V. harveyi UM1503||+||+||+||−|
|V. harveyi UMBB7||+||+||+||−|
|V. metschnikovii UM4368||+||+||+||+|
|V. mimicus ATCC33653||+||+||+||+|
|V. mimicus UM4053||+||+||+||+|
|V. mimicus UM4194||+||+||+||+|
|V. mimicus UM4198||+||+||+||+|
|V. mimicus UM4205||+||+||+||+|
|V. mimicus UM4208||+||+||+||+|
|V. mimicus UM6812||+||+||+||−|
|V. mimicus UMRB653L||+||+||−||−|
|V. natriegens UM4239||+||+||−||+|
|Vibrio nereis UM4231||+||+||+||−|
|V. parahaemolyticus ATCC15338||−||−||−||−|
|V. parahaemolyticus ATCC17802||+||+||+||+|
|Vibrio tubiashii ATCC19109||−||−||+||−|
|Vibrio vulnificus UMC7184||+||+||+||+|
|V. vulnificus UME4125||+||+||−||−|
|V. vulnificus UM543||−||−||+||−|
|Total positive/total tested||32/53d||37/58||33/58||21/58|
2.2Bacterial counts from Chesapeake Bay water samples
Water samples were collected from the Chesapeake Bay during March and August 1995, and November 1997. The following bacterial counts were determined in water samples collected during August 1995: total direct counts (TDC) by acridine orange staining and epifluorescence microscopy , nucleoid-containing cells (NUCC) , direct viable counts (DVC) , and colony forming units (CFU) on 1/10 strength Trypticase Soy Agar (TSA) (Difco) supplemented with 1% NaCl (wt./vol.).
2.3Extraction of DNA
Genomic DNA was extracted by a modification of a standard method incorporating a hexadecyl trimethyl ammonium bromide (CTAB) treatment step . Briefly, strains grown as described above in 100 ml LB were harvested by centrifugation at 4000×g. The resulting cell pellets were resuspended in 9.5 ml Tris–EDTA (TE) buffer, 0.5 ml 10% sodium dodecyl sulfate (SDS), and 50 μl 20 μg ml−1 proteinase K, and mixed well. This mixture was incubated for 1 h at 37°C. NaCl (1.8 ml of a 5 M solution) and 1.5 ml of 10% CTAB in 0.7 M NaCl was added and the mixture incubated at 65°C for 20 min. DNA was purified by a standard phenol-chloroform extraction , the aqueous phase was transferred to a fresh tube and the DNA precipitated by addition of 0.6 vol. of isopropanol. The DNA was washed with ethanol (70% (v/v)) and dried under vacuum. After overnight resuspension in TE at 4°C, the DNA solutions were treated with RNase A (Boehringer-Mannheim), re-precipitated, and concentration determined by UV spectroscopy.
Primers were designed for PCR amplification of chiA gene fragments by alignment and selection of highly conserved regions of several chiA sequences from the GenBank database (organisms and GenBank accession numbers are: Serratia marcescens X03657, Z36294; Alteromonas sp., D13762; Bacillus circulans, M57601 and Aeromonas caviae, U09139). The primers, 5′-GATATCGACTGGGAGTTCCC-3′ (forward; corresponding to nucleotide positions 931–950 of S. marcescens X03657) and 5′-CATAGAAGTCGTAGGTCATC-3′ (reverse: positions 1180–1161) flanked a 225 bp fragment. Oligonucleotides were synthesized using an Applied Biosystems 380A Automated DNA Synthesizer, purified, lyophilized, resuspended in ultrapure water, and concentrations determined by UV spectrophotometry. The following reagents were used for chiA amplification: 50 ng DNA from V. harveyi ATCC14216; 10 pmol of each primer; 100 mM of each deoxynucleotide triphosphate; 2.5 units Taq polymerase (Perkin-Elmer); and 1×Taq polymerase buffer in a final volume of 25 μl. PCR reaction conditions were: 94°C for 4 min; 35 cycles of denaturation at 92°C; annealing at 58°C; and extension at 72°C for 1 min each, followed by 7 min extension at 72°C. PCR products were visualized by agarose gel electrophoresis.
2.5Probe preparation and labeling
A chiA probe for colony and dot-blot hybridizations was prepared by radiolabeling the chiA PCR amplicon from V. harveyi. The 225 bp fragment was resolved by agarose gel electrophoresis, excised from the gel, and purified using the Wizard PCR Preps DNA Purification System (Promega), following the manufacturer's instructions. Purified DNA was labeled with (α-32P)-dCTP using a nick translation kit (Boehringer Mannheim). V. cholerae UM400 and Vibrio parahaemolyticus ATCC15338 were included in dot-blots and colony hybridizations as negative controls.
Water samples were collected at four depths from four different sampling locations in the Chesapeake Bay during March and August 1995. One surface sample was collected offshore at St. Michael, MD, USA, during November 1997. Four to five replicates of appropriate dilutions of these samples were spread plated on 1/10 strength TSA plates. Plates were incubated at 25°C until colonies formed, usually after about 30 h. Colony forming units were enumerated and plates containing approximately 150 to 200 colonies were selected for colony blot experiments. In some cases, colony hybridizations were done on ordered arrays of colonies which had been previously purified from the initial isolation plates, namely, 650 colonies isolated from samples collected during March 1995. Fifty of these isolates were transferred to each of 13 plates. Colonies were transferred to Magna nylon membranes (Micron Separation Inc). Membranes were prepared as follows: lysis (10% (w/v) SDS for 3 min); denaturation (0.5 M NaOH and 1.5 M NaCl for 5 min); neutralization (0.5 M Tris–HCl, pH 8.0 and 1.5 M NaCl for 5 min); and rinse (2×SSC for 1 min). Processed membranes were baked at 80°C under vacuum for 2 h. Membranes were placed in prehybridization solution (5×SSC, 10×Denhardt's solution  and 500 μg salmon sperm DNA per ml) at 55°C. After prehybridization for 2 h, hybridization was carried out at 55°C overnight. Membranes were washed twice in 0.15 M NaCl–0.015 M sodium citrate (SSC), 0.1% SDS for 15 min at room temperature, twice with 1×SSC, 0.5% SDS for 20 min at 37°C, twice with 1×SSC, 0.5% SDS for 30 min at 55°C, and once with 1×SSC, 0.5% SDS at 65°C for 1 h and exposed to X-Omat AR film at −70°C for 1–8 h. Dot-blots were performed on pure DNA samples as described elsewhere , using the same wash conditions as for colony hybridizations.
The ability of environmental isolates and reference strains to utilize different forms of chitin was determined by assessing growth in a minimal medium containing KH2PO4 (0.5 g−1), MgSO4·7H2O (0.2 g−1) and FeSO4·7H2O (0.01 g−1). Crabshell chitin (Sigma), N-acetylglucosamine (NAG) (Sigma), and diatom chitin (a gift from Dr. Alan Place) were added individually to the minimal medium to a final concentration of 2.5 g l−1 and the medium was autoclaved. Strains to be tested were grown in Tryptic Soy Broth (TSB) (Difco) supplemented with 1% NaCl with shaking for 18 h at 30°C, harvested by centrifugation, and washed in 1% saline solution. The resulting cell pellets were resuspended in 1% saline and 50 μl (ca. 2×106 cells per ml) of these suspensions were added to tubes supplemented with each form of chitin. These cultures were incubated at 30°C with shaking. Growth was monitored visually for 35 days, after which the presence of culturable cells was confirmed by growth on TSA plates. V. cholerae UM400 and V. parahaemolyticus ATCC15338 were included as negative control strains.
2.8Cloning, sequencing and phylogenetic analysis of chiA amplicons
Amplification products of the predicted size of ca. 225 bp, obtained using the chiA primers, were cloned and sequenced. PCR products were purified from low melting agarose gels using the Wizard PCR Preps DNA Purification System. Purified DNA fragments were cloned into the pGem-T Easy vector (Promega) following manufacturer's instructions. White colonies were selected and plasmids prepared using the Wizard Miniprep DNA Purification Kit (Promega). After confirming the presence of inserts of ca. 225 bp by EcoRI digestion, both strands of recombinant plasmids were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Kit and an ABI 377 (Perkin-Elmer) automated DNA sequencer. Two primers flanking the multiple cloning site of the pGem-T Easy vector, namely pGTf (5′-TACGACTCACTATAGGGCGA-3′) and pGTr (5′-ACTCAAGCTATGCATCCAACGC-3′), were used for thermal cycling sequencing reactions. Sequences were aligned using the PILEUP program in the University of Wisconsin Genetics Computer Group software package. After manual adjustments, the alignment was used in phylogenetic analyses. Phylogenetic trees were inferred using the neighbor-joining ), Fitch–Margoliash  and maximum parsimony methods . Evolutionary distance matrices were generated as described elsewhere  for amino acid and nucleotide sequences , respectively. The PHYLIP package  was used for all analyses. The resultant unrooted tree topologies were evaluated using bootstrap analyses  of the neighbor-joining method, based on 1000 resamplings.
The chiA sequences determined in this study were deposited in the GenBank database under accession numbers AF059494–AF059504. Strains and corresponding accession numbers are listed in the legend to Fig. 1.
3.1Detection of chiA genes and chitinolytic activity in reference strains
In over half of the 53 reference strains examined in this study, a fragment of the appropriate size (ca. 225 bp) was amplified by chiA gene-specific primers (Table 1). These strains were designated as chiA gene-positive (Table 1). Results were confirmed by probing dot-blots of DNA from all reference strains with 32P-labeled chiA DNA amplified from V. harveyi ATCC14216 and in all cases results were consistent with those obtained by PCR detection of the chiA gene.
Growth of reference strains on crabshell chitin, diatom chitin, and NAG, indicated by visible turbidity, is indicated in Table 1. Only 13 of 37 strains which were chiA-positive by PCR detection of the chiA gene gave growth on all three chitin substrates. However, all strains positive for chiA by PCR grew on the crabshell chitin substrate. Utilization of NAG and/or diatom chitin was not shown by 25 of these chiA-positive strains. In addition, 13 of 21 strains which were negative for the presence of chiA by PCR and dot-blot hybridization were positive for growth on NAG and/or diatom chitin.
3.2Enumeration of chiA-positive bacteria in Chesapeake Bay water samples
Pure cultures of bacteria isolated from water samples collected from the surface and near the bottom at two locations in Chesapeake Bay during March 1995 (stations 724 and 858) were tested for presence of the chiA gene using the chiA probe generated by PCR from V. harveyi template DNA (Table 2). Complete microbiological counts for these stations are reported in Ramaiah et al. . Of 650 isolates tested, ca. 29% of the isolates were chiA-positive. These hybridization results were confirmed by PCR analysis of five chiA probe-positive and five probe-negative colonies. The chiA probe was also used for direct enumeration on isolation plates of colonies with the chiA genotype. For each sample, ca. 400 colonies were tested. A high proportion (average 21%) of the culturable bacterial population was found to be chiA-positive (Table 2). There was no statistically significant variation in the proportions of culturable chiA-positive bacteria with depth (P>0.55) or between stations (P>0.57). The chiA-positive bacteria ranged from 5 to 41% of the culturable bacteria present in the water samples. To confirm these results, 10 probe-positive and probe-negative isolates were examined by PCR and by dot-blotting techniques; results were consistent with those obtained using the chiA gene probe. Five of the probe-positive isolates were tested for utilization of the chitin substrates and results are included in Table 1. Five probe-negative isolates were negative for utilization of crabshell chitin, diatom chitin and NAG.
|Station||Location||Sampling depth||Culturable cell count (×103 ml−1)||Proportionb of chiA-positive cells|
|Averages of all counts|
The complete sequence of the chiA gene from V. harveyi strain BB7 was recently reported  and the partial chiA sequence of V. harveyi ATCC14216 obtained in this study is identical to the corresponding region of the V. harveyi strain BB7 chiA gene. Eleven chiA sequences determined in this study were aligned with nine published sequences using the PILEUP program, followed by manual adjustments. The resultant multiple alignment is given in Fig. 1. Phylogenetic trees were inferred, using both chiA amino acid (Fig. 2A) and nucleotide sequence data (results not shown). In addition, the corresponding phylogenetic tree, based on 16S rRNA, was generated using published data for comparative analysis (Fig. 2B), as 16S rRNA sequences of most test strains are available in the GenBank database. The phylogenies were similar for both chiA amino acid and nucleotide trees. The phylogenetic position of Plesiomonas shigelloides is somewhat unstable, as the branching points in both trees are different and supported by very low bootstrap values (23 and 37 for DNA and protein trees, respectively). Similarly, the placements of Vibrio fisheri are not in agreement in DNA and protein trees. The 16S rRNA tree was remarkably consistent as the same tree topology was recovered from all three tree-making algorithms and supported by high bootstrap values throughout all branching points (Fig. 2B).
From an ecological point of view, there is great interest in the enumeration of chitinoclastic microorganisms by rapid, reliable detection techniques [33,34]. In the present study, chiA gene-specific PCR primers and a chiA gene probe were developed and their specificity verified using a large assemblage of reference strains. The chiA fragments amplified by PCR from representative strains were sequenced to confirm their specificity. Chitinolytic activity of the reference strains, in general, was consistent with activities reported in the literature. These results, together with our detection of chiA-positive bacteria in environmental water samples, confirmed that the PCR-amplified chiA probe was useful for easy and unambiguous identification of chitinoclastic genotypes present in environmental samples.
A high proportion (average 21%) of the culturable bacteria isolated from Chesapeake Bay water samples was positive for the presence of chiA gene sequences. Up to 30% of total culturable heterotrophic bacteria have previously been reported to be chitinoclastic on phenotypic examination . In a recent study on chitinases from uncultured marine microorganisms, the estimates of the percentages of chitin degraders for the estuarine and coastal communities were 5.5 and 0.12%, respectively . Chitinase activity can be much higher on particles than in the water column . The presence of a high concentration of particulate material in the Chesapeake Bay may be a factor accounting for the high proportion of chiA-positive bacteria in water samples from the Bay. A number of environmental parameters are understood to influence the distribution and abundance of chitinoclastic bacteria in the marine environment [9,37]. Concentrations of chitin in the water column in Delaware Bay have been shown to be low, ranging from 4 to 21 μg l−1 but low concentrations of chitin in the water column may be due to rapid cycling of this important carbon and nitrogen source, rather than indicating low chitin production rates . In the York River Estuary, Virginia, USA, chitin degradation was shown to occur very rapidly and it was concluded that water column bacteria may be more important in this regard than previously acknowledged . The high counts of chiA-positive bacteria obtained using a molecular probe in this study support this conclusion. Since chitin is such an abundant polymer in the marine environment, it is not surprising that a high proportion of heterotrophic marine bacteria has evolved the ability to utilize this carbon and nitrogen source. All five chiA-positive isolates from the Chesapeake Bay were able to utilize at least one chitin substrate.
Rapid detection of chitinoclastic bacteria from natural sources may also be useful in isolation of bacteria for screening for antimicrobial or other novel compounds. A high correlation between chitinolysis and production of bioactive compounds has been reported . In addition, nearly every Streptomyces species is chitinolytic [40,41] and this group of bacteria is a particularly important group for production of bioactive compounds. A large number of chitinoclastic marine actinomycetes were previously found to suppress growth of bacteria and fungi . A detailed analysis of the putative catalytic region from bacterial chitinases grouped chitinases from actinomycetes into three of five groups . Two of six actinomycetes included in this analysis grouped with V. harveyi, suggesting that the chiA probe developed in this study may be useful for selective isolation of some chiA-positive actinomycetes for use in natural products screening programs.
Although the sequences of chiA genes from different species of bacteria show significant divergence, the primers we designed were targeted to highly conserved regions of the chiA gene and were able to detect chiA in a wide range of bacterial species. All strains which were positive for growth on crabshell chitin (α form) were found to possess chiA. However, some strains which grew on diatom chitin (β form) or on NAG were found not to possess a chiA gene. Depending on the purity of the chitin, growth may occur on trace nutrients, other than chitin. However, diatom chitin is generally regarded as pure and free of associated proteins and other compounds that may contaminate crab chitin. It is therefore likely that in these strains, enzymes for chitin degradation are encoded by chitinase genes other than chiA. It is also possible that these strains possess a chiA gene which is not detectable by our primers and probe but this is less likely since several marine bacteria have been shown to possess multiple chi genes, apparently synthesized for efficient utilization of different forms of chitin . The approach used in this study is applicable to the development of PCR primers and probes for detection of additional chitinases, which could further increase the proportion of heterotrophic marine bacteria in the water column shown to be chitinoclastic.
Sequence analysis confirmed that the PCR amplicons obtained using our primers are indeed chiA homologues. Similar phylogenetic trees were obtained from the chiA gene sequence and ChiA protein sequence analysis, as is generally expected. The phylogenies based on chiA and 16S rRNA gene sequences are in close agreement. V. fisheri was an exception and did not group with other vibrios in the chiA trees. The chiA gene sequence of the Chesapeake Bay isolate 11d was identical to that of Vibrio natriegens, indicating that this isolate may be a V. natriegens strain. However, isolate 6d was consistently placed outside of the clade corresponding to the gamma-proteobacterial division in both chiA trees (Fig. 2A,B). In addition, an insertion of eight amino acids starting from position 331 (S. marcescens numbering) was found in the isolate 6d. It was, therefore, impossible to deduce the identity of isolate 6d from chiA sequence data.
It is interesting to note that not all strains of V. cholerae were capable of growth on crabshell chitin nor were positive for chiA. However, most V. cholerae strains grew on NAG and/or diatom chitin. The distribution and survival of V. cholerae in marine ecosystems have been of great interest for decades  and attachment to surfaces of zooplankton, in particular the copepods, is an important reservoir of V. cholerae in the environment . Surface membrane proteins in V. cholerae able to bind chitin particles were recently identified . The relationship between chitinase utilization, presence of chi genes, and pathogenicity in V. cholerae warrants further investigation.
Our approach for detection of chiA-positive bacteria has proved useful for examining the incidence and distribution of this genotype in the natural environment. Clearly, molecular techniques will be required in the future to investigate the ecology of chitinoclastic microorganisms, exemplified by the recent work by Cottrell et al.  in which selected chitinase genes in uncultured as well as cultured marine bacteria were studied.
We thank Anwar Huq and Chris Grim for helpful discussions. The crew of R/V Cape Henlopen and Drs. Eric Wommack, Diane Stoecker and Wayne Coats provided valuable assistance in sample collection. N.R. is grateful to the Department of Biotechnology (Ministry of Science and Technology), New Delhi, for an Overseas Associateship award, to the American Society for Microbiology for an UNESCO-ASM Travel Award, and to Dr. D. Chandramohan at the NIO for encouragement and support.
- Austin, B. (1988) Marine Microbiology. Cambridge University Press, Cambridge.
- 1971) Chitinous structures. Compr. Biochem. 26, 595–632.(
- 1973) Microbial aspects of penaeid shrimp digestion. Proc. Gulf Caribbean Fish. Inst. 26, 81–92., (
- 1977) Rates of chitin degradation in an estuarine environment. J. Oceanogr. Soc. Jpn. 33, 328–334., (
- 1968) Studies on chitin decomposing bacteria present in digestive tracts of aquatic animals. III. Formation of organic acids. Bull. Jpn. Soc. Sci. Fish. 34, 1141–1146., (
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
- Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1987) Current Protocols in Molecular Biology. Wiley, Cambridge, MA.
- Dayhoff, M.O. (1978) Atlas of protein sequence and structure. The National Biomedical Research Foundation, Washington, DC.
- Jukes, T.H. and Cantor, C.R. (1969) Evolution of protein molecules. In: Mammalian Protein Metabolism (Munro, H.N., Ed.), pp. 21–132. Academic Press, New York.
- Felsenstein, J. (1993) Department of Genetics, University of Washington, Seattle, WA.
- Ramaiah, N., Chun, J., Ravel, J., Straube, W.L., Hill, R.T. and Colwell, R.R. (1999) Detection of luciferase gene sequences in ‘visibly non-luminous’ bacteria from the Chesapeake Bay. FEMS Microbiol. Ecol., in press.
- 1994) Distribution of heterotrophic bacteria in seawater near Taiwan and application of a proteolytic and chitinolytic isolate. J. Fish. Soc. Taiwan 21, 197–204., (
- 1991) Distribution and hydrolytic enzyme activities of aerobic, heterotrophic bacteria isolated from grass prawn, Penaeus monodon. J. Fish. Soc. Taiwan 18, 301–310., , , (
- Kutzner, H.J. (1981) The family Streptomycetaceae. In: The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria, pp. 2028–2090. Springer, Berlin.