Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the mannose/glucose permease.

We have previously reported that the chitin catabolic cascade in Vibrio furnissii involves multiple signal transducing systems, and that mono- and disaccharide chemoreceptors/transporters are essential components of some of these systems. This and the accompanying papers (Bouma, C. L., and Roseman, S. (1996) J. Biol. Chem 271, 33457-33467; Keyhani, N. O., Wang, L.-X., Lee, Y. C., and Roseman, S. (1996) J. Biol. Chem. 271, 33409-33413) describe some of the sugar transporters. A 13-kilobase pair fragment of V. furnissii DNA was found to impart a Glc+, Man+ phenotype to Escherichia coli ptsG ptsM mutants, and encodes the mannose transporter, ptsM, of the phosphoenolpyruvate:glycose phosphotransferase system. Unlike the E. coli mannose permease, V. furnissii IIMan is inactive with GlcNAc and Fru, and is encoded by four genes rather than three. The gene order is manXYZW, where the product of manY corresponds to IIPMan, manZ to the mannose receptor IIBMan, and manX and manW to the single E. coli gene, manX (which encodes IIIMan, viz. IIAMan). Thus, in V. furnissii, the E. coli manX equivalent comprises two genes, which are separated in the genome by two other genes of the ptsM complex. Two additional open reading frames were detected in the V. furnissii DNA fragment. One encodes a GlcNAc-6-P deacetylase, and the other is similar to aldolase.

II Man (2,3). Indeed, in screening V. furnissii genomic libraries in E. coli, a V. furnissii II Man equivalent was detected because it conferred a Glc ϩ phenotype to E. coli ptsG ptsM mutants. We report here that V. furnissii ptsM differs from its E. coli homolog in the number of polypeptides required for its function, the gene order, and in its substrate specificity.
The cloned V. furnissii DNA fragment contained two additional open reading frames. The deduced amino acid sequences of these open reading frames indicate that one encodes a Glc-NAc-6-P deacetylase, and the other a Fru-1,6-diP aldolase.  (6). The genotypes of strains and plasmids not given here are shown in Table I or Table III. The plasmid pCAR-3 was a gift from Dr. C. A. Roessner (Department of Medical Biochemistry, Texas A&M College of Medicine, College Station, TX); E. coli LR2-175 was a gift from Dr. J. Lengeler (Universitat Osnabruck, Fachbereich Biologie/Chemie, Osnabruck, Germany); IBPC531 was a gift from Dr. J. Plumbridge (Institute de Biologie Physico-chimique, URA1139, Paris, France). For the preparation of toluene-permeabilized cells, E. coli transformants were grown in M9 medium (10) supplemented with 0.1% casamino acids, 0.5% Man, and the appropriate antibiotic to A 500 ϭ 0.8. The accompanying article (1) describes culture conditions for transformations, transductions, plasmid preparations, sugar fermentation, and sugar phosphorylation assays.

Biochemicals and Molecular Biology Reagents-Restriction
Construction and Screening of V. furnissii DNA Libraries-Genomic libraries of BamHI and HindIII fragments of V. furnissii 1514 DNA in the vector pBR322 were constructed and screened for plasmids conferring a Glc ϩ phenotype to E. coli SR423 or SR425 as reported in the accompanying paper.
DNA Sequencing and Analysis-One strand of the SalI fragment of V. furnissii DNA contained in the plasmid p3H1S was sequenced by the Genetics Core Facility, Johns Hopkins Medical Institutions, Baltimore, MD. The complementary strand was sequenced by the dideoxy chaintermination method (11) with Sequenase T7 polymerase (U. S. Biochemical Corp.) as described in the accompanying article (1). The V. furnissii fda gene is truncated in p3H1S, and its sequence was therefore determined from p3H1. Primers were designed as needed while sequencing, and were synthesized and purified according to the manufacturer's recommendations (Applied Biosystems, Inc). The analysis of DNA and amino acid sequences was carried out with the GCG sequence analysis package (Version 7, Genetics Computer Group, Madison, WI). The data bases searched for nucleotide and amino acid sequence similarities were: GenBank™ Release 79 and Swiss Protein Release 26.
Predicted amino acid sequences were aligned using CLUSTAL W version 1.5; similarities were shown by shading using BOXSHADE/DOS 2.7, kindly performed by Michael Cleveland.
Assay for Sensitivity-E. coli transformants harboring plasmid clones of the V. furnissii man operon were scored for sensitivity to vir by the cross-streak assay (6). Recipient cells were grown to A 500 ϭ 0.8 -1.0 with good aeration in LB supplemented with 0.2% maltose, 10 mM MgSO 4 , and the appropriate antibiotic. Complete lysis of the test strain was indicative of sensitivity (the ability of vir DNA to penetrate the inner membrane via Enzyme II Man ).
In  (1,13), and membranes prepared as described (14). PTS Enzyme II activity was measured as reported (14), except that 25 M HPr was used and III Glc was omitted from the Enzyme II Man assay mixture. Specific activities are expressed as nanomoles of sugar phosphate formed ϫ min Ϫ1 ϫ mg of membrane protein Ϫ1 .
Sugar Phosphorylation by Toluene-permeabilized Cells-E. coli transformants harboring recombinant plasmids and V. furnissii SR1514 were grown, harvested, washed, and toluene-permeabilized (13). Sugar phosphorylation was measured as described. Specific activities are expressed as nanomoles of sugar phosphate formed ϫ min Ϫ1 ϫ mg of cell protein Ϫ1 .
c Fermentation was scored on Maconkey agar with 1% sugar and the appropriate antibiotic. ϩ, dark red colonies; ϩ/Ϫ, red colonies; Ϫ, light pink/white colonies; NT, not tested. d Tests of sensitivity were conducted, in SR425 only, by the cross-streak assay (6) as described under "Materials and Methods." E. coli SK1592 (ManX ϩ Y ϩ Z ϩ ), transformed with pBR322, was used as a positive control. synthesized and purified as described above. Nucleotide numbers refer to Fig. 3, and sequences are given in the 5Ј to 3Ј direction: primer A, nt 1-23; primer B, nt 1658 -1643 with a 5Ј EcoRI site; primer C, 1562-1583 with a 5Ј PvuI site; primer D, nt 3030 -3009 with a 5Ј PstI site; primer E, nt 2545-2525 with a 5Ј EcoRI site; primer F, nt 2482-2503 with a 5Ј PvuI site; and primer G, nt 903-880 with a 5Ј EcoRI site. Amplification conditions are given in the accompanying article (1). The PCR products were ethanol precipitated, digested with the appropriate restriction enzymes and ligated into the vectors pUC19 or pBR322. The following restriction digests and ligations were carried out: the PCR products of primers A ϩ B, primers A ϩ E, and primers A ϩ G were digested with HindIII and EcoRI and ligated into pUC19 (Amp R ); the PCR products of primers C ϩ D and primers F ϩ D were digested with PvuI and PstI and ligated into pBR322 (Tet R ). Subsequent testing for complementation of Man fermentation and sensitivity was accomplished by co-transformation of the various Man subclones into E. coli ptsM mutants. Transformants resistant to both antibiotics were selected and maintained on media containing both ampicillin (50 g ml Ϫ1 ) and tetracycline (15 g ml Ϫ1 ), and the presence of both plasmids was confirmed by restriction enzyme digestion of plasmid minipreps.

RESULTS
Complementation of E. coli ptsG ptsM mutants-Two libraries of V. furnissii DNA, in the HindIII and BamHI sites of pBR322, were screened for plasmids that complemented the Glc fermentation defect in E. coli SR423. Four plasmids with non-homologous fragments of V. furnissii DNA were isolated. One clone, designated p3H1, containing 13 kb of V. furnissii DNA, is described in this report; the characteristics of two additional clones are presented in an accompanying paper (1). The fourth clone encodes a glucose transporter that will be described elsewhere.
Restriction fragments of the plasmid p3H1 were subcloned to determine the minimum size of V. furnissii DNA that would complement the Glc Ϫ defect in E. coli SR423, and a 5.1-kb SalI fragment met this requirement. The restriction maps of p3H1 and the subclone p3H1S are shown in Fig. 1.
The phenotypes conferred by these plasmids were assessed in different E. coli pts mutants. As shown in Table I, the plasmids p3H1 and p3H1S restored the Glc ϩ , Man ϩ phenotype to ptsG ptsM mutants containing the other required components for uptake and catabolism of these sugars. The plasmids also complemented the Glc and Man fermentation defects in the strains ZSC113, SR423, and JM1100 (data not shown). That the plasmids conferred Glc ϩ , Man Ϫ phenotypes to LR2-175 and its derivatives (Table I) was expected, given the phosphomannose isomerase mutation in this strain. The gene products of both plasmids required Enzyme I and HPr for fermentation, but were III Glc -independent.
The results summarized above therefore show that the cloned V. furnissii II Man complex cross-reacts in vivo with E. coli phospho-HPr. As shown below, this is also true in vitro.
One of the characteristics of the E. coli ptsM gene complex is that it encodes the membrane protein Pel, or IIP Man . This protein is required for penetration of the inner membrane by phage DNA. Phage sensitivity assays conducted with SR425 transformants, however, showed that the clones containing V. furnissii DNA did not permit infection. The phenotypic behavior of E. coli cells harboring the cloned V. furnissii genes differed from those carrying pCAR-3, a plasmid carrying the complete E. coli ptsM locus (manXYZ). Despite allowing GlcNAc fermentation comparable to pCAR-3 transformants, the presumptive V. furnissii ptsM clones did not restore growth on minimal media with GlcNAc or Fru as the sole source of carbon. Transformation with the plasmid pCAR-3 allowed growth on minimal Man and GlcNAc, and weak growth on Fru (data not shown).
Sugar Phosphorylation in Vitro-The phenotype conferred upon E. coli ptsG ptsM mutants by the V. furnissii p3H1S is compared to the results of in vitro sugar phosphorylation assays in Table II. Glc phosphorylation by membranes from p3H1S transformants was 3-25-fold greater than obtained with controls, membranes isolated from strains transformed with the vector. Similarly, Man phosphorylation by p3H1S transformant membranes was 4 -10-fold greater than background levels. The III Man (viz. IIAB Man ) subunit of the E. coli Enzyme II Man complex is soluble (15,16), and we hypothesized that the low specific activities observed with p3H1S transformants might be due to the loss of the III Man homolog during membrane preparation. However, neither the addition of the soluble fraction from an E. coli III Man overproducing strain nor the addition of the soluble fraction from Man-grown V. furnissii 1514 stimulated Glc or Man phosphorylation by membranes from p3H1S transformants (data not shown). More convincing results were obtained with toluenized cell preparations (Table  II), where Man phosphorylation was from 10-to 50-fold higher than the background level. The phosphorylation data therefore   correlate with the phenotypic results presented in Table I.
While mannose and glucose were clearly phosphorylated by both the membrane and toluenized cell preparations, the activities were low compared to preparations obtained from cells transformed with the plasmid pCAR-3, which carries the E. coli mannose operon, i.e. the genes manXYZ. There are at least two possible explanations for these results; (a) cells transformed with pCAR-3 express more ptsM proteins than do cells transformed with p3H1S, or (b) the Vibrio ptsM proteins do not interact efficiently with E. coli phospho-HPr.
Although Fru and GlcNAc are substrates of E. coli II Man , phosphorylation of these compounds by membranes from p3H1S transformants was not detectable (data not shown). Membranes from the transformants were also tested with 2-deoxyglucose and methyl ␣-D-glucopyranoside. These analogues are commonly used with E. coli cells and membranes to distinguish between ptsM and ptsG, respectively (17). No phosphorylation of either compound was detected, although 2-deoxyglucose is an excellent substrate for E. coli II Man .
In Vitro Translation of p3H1S-To assess the number and molecular weights of polypeptides encoded by p3H1S, the plasmid was translated in a prokaryotic in vitro system (Fig. 2). In addition to the vector-encoded ␤-lactamase, four peptides were translated from p3H1S, with apparent molecular masses of 30.5, 26.5, 17.5, and 16.5 kDa. An additional band was observed with an apparent molecular mass of 42 kDa. The results in Fig. 2 are discussed further below.
DNA Sequence Analysis-The 4.6-kb V. furnissii DNA fragment of p3H1S was sequenced (11) by the dideoxy chain-termination method. Four open reading frames with identity to the subunits of E. coli Enzyme II Man were identified. To be consistent with the established nomenclature of the Man PTS (4, 18), we have designated these genes manX, manY, manZ, and manW, respectively. A fifth open reading frame (manD) overlapping the terminal 11 nucleotides of manW, encodes a polypeptide with GlcNAc-6-P deacetylase activity. Finally, a sixth open reading frame, which overlaps the last 10 nucleotides of manD, resembles several bacterial Fru 1,6-P aldolases and was designated manF. Fig. 3 shows that these six open reading frames are closely linked and may be co-transcribed. The termination codon of manX is 15 nucleotides from the initiation codon of manY, the putative ribosomal binding site and initiation codon of manZ are within manY, and manW begins 57 nucleotides from the termination codon of manZ. A putative ribosomal binding site was identified for each coding region (Fig. 3), based upon identity with the E. coli ribosomal binding site (19). A potential promoter (20) for the man operon is also highlighted in Fig. 3. An 8-nt inverted repeat sequence is located between manZ and manW. Following manF are two GC-rich regions of dyad symmetry preceding a series of T residues, a sequence that could function as a transcriptional terminator in the mRNA.
Identification of V. furnissii Enzyme II Man Subunits-In or-der to correlate the V. furnissii man genes with the polypeptides observed in Fig. 2, the man genes were amplified from genomic DNA, individually and in various combinations, and were analyzed by in vitro translation. The plasmids were also used in attempts to complement E. coli ptsM mutants of defined genetic defects. Table III shows that Man fermentation was possible by the E. coli ptsM mutants only when all four V. furnissii Enzyme II Man subunits were present. V. furnissii ManW and ManX, the homologs of E. coli III Man , did not complement an E. coli III Man mutant whether the two V. furnissii subunits were present individually or were co-transformed.
The clones of the V. furnissii Enzyme II Man subunits were used as templates for in vitro translation in order to assign the observed products to their respective open reading frames. The results (Fig. 2) indicate that ManY (27 kDa) corresponds to the 26-kDa labeled polypeptide and ManZ (31.7 kDa) to the 30.5-kDa labeled peptide. ManW (15.9 kDa) and ManX (17.2 kDa), however, migrate as 17.5-and 16.5-kDa proteins, respectively. The apparent molecular masses of the translation products agreed reasonably well with the predicted values, except that the product of manW migrated somewhat more slowly than expected.
Identification of GlcNAc-6-P Deacetylase and Fructose 1,6-BisP Aldolase-The faint, 42-kDa band observed in p3H1S transcription/translation reactions corresponds closely to the predicted molecular mass (43 kDa) of the ManD gene product. Only the plasmid p3H1S directed the in vitro synthesis of this polypeptide. Furthermore, a functional GlcNAc-6-P deacetylase is encoded by p3H1 and p3H1S, since both plasmids com-  Table I Table I). The deduced amino acid sequence of manD is 29% identical to E. coli GlcNAc-6-P deacetylase (Fig. 4).
A gene encoding a potential V. furnissii Fru 1,6-bisP aldolase overlaps manD (Fig. 3), and we have named this gene manF. The deduced amino acid sequence of this protein shares up to 41% identical residues with Class II aldolases from E. coli (21), Rhodobacter sphaeroides (22), and Bacillus subtilis (23). More interestingly, its sequence is most similar to GatY, which is encoded by the E. coli gat operon and reportedly carries ketose bisphosphate aldolase activity (24). An alignment of ManF with E. coli GatY and B. subtilis aldolase is given in Fig. 5. Whether V. furnissii manF directs the synthesis of a functional aldolase was not determined in this study. DISCUSSION We have proposed that (a) chemotaxis to Glc and trehalose may be the mechanism by which marine (and terrestrial?) bacteria locate chitin-producing organisms (13), and (b) chemotaxis to and transport of GlcNAc are essential for functioning of the chitin catabolic cascade in V. furnissii (9). An accompanying paper (1) describes the cloning and characterization of a Glc and a GlcNAc transporter from this organism. Both are PTS sugars in V. furnissii, and the respective Enzyme II complexes serve as the sugar receptors in both chemotaxis and uptake. However, bacteria often use more than one system for chemotaxis/transport to a given sugar. The II Man complex of E. coli, for example, recognizes both Glc and GlcNAc. To more fully understand the physiology of V. furnissii therefore required an extensive search for alternative chemotaxis/transporters for Glc and GlcNAc. The strategy that we followed was to screen libraries of V. furnissii DNA in E. coli Glc Ϫ mutants, since Glc is transported by the largest number of systems (both PTS and non-PTS transporters) in the enteric bacteria. We report here that such screenings led to the molecular cloning of the V. furnissii ptsM complex.
The Enzyme II complexes of the PTS comprise from one to as many as four individually coded polypeptide chains (for re-views, see Refs. 25 and 26). The most complex are the B. subtilis fructose or levulose (27) and Klebsiella pneumoniae Sor (L-sorbose) Enzyme II complexes (28,29), which consist of four proteins each. The sequences of these proteins are similar to the three proteins of the E. coli II Man complex. The latter is encoded by three adjacent genes (4, 15) designated manX (encodes III Man , also called IIAB Man ), manY (encodes IIP Man ), and manZ (encodes IIB Man ). 2 The protein IIP Man (and also possibly IIB Man ) is required for phage DNA penetration of the E. coli inner membrane (4). However, the V. furnissii homolog does not permit penetration of the cytoplasmic membrane by phage DNA. E. coli II Man transfers the phosphoryl group from phospho-HPr to Man, Glc, 2-deoxyglucose, Fru, GlcNAc, GlcNH 2 , and other sugars, concomitant with their translocation (25,29). By sharp contrast, the V. furnissii II Man complex apparently phosphorylates only Man and Glc.
Nucleotide sequence analysis of the cloned V. furnissii DNA revealed six closely linked open reading frames (Fig. 3). The first four showed identity with the subunits of the E. coli Man permease. The other two encoded proteins similar to GlcNAc-6-P deacetylase and fructose 1,6-bisP aldolase.
Four separate proteins were encoded by the first four open reading frames in the plasmid p3H1S. All four proteins were required for sugar phosphorylation, and they functioned, both in vivo and in vitro, with E. coli phospho-HPr as the phosphoryl donor. By marked contrast, individual subunits of the V. furnissii permease were unable to substitute for their E. coli II Man homologs, i.e. a functional II Man complex was observed only when the transformants expressed the three E. coli or the four V. furnissii proteins. Apparently there is insufficient homology between the V. furnissii and E. coli permeases to allow intersubunit complementation. It is, however, important to note that the V. furnissii proteins formed functional II Man complexes when transcribed from different plasmids. From this it appears that the integral membrane components of the V. furnissii II Man complex properly fold and recognize one another after being separately expressed. It will be interesting to determine whether this association takes place before or after insertion into the membrane.
Enzyme II complexes comprise three functional domains: a hydrophilic domain, which possesses the first phosphorylation site and accepts the phosphoryl group from HPr; a second hydrophilic domain containing another phosphorylation site; and a hydrophobic transmembrane domain, which binds and transports the sugar. The domains may be in a single or in separate proteins. In the E. coli II Man complex, there are two transmembrane domains, IIB Man and IIP Man , and a hydrophilic subunit, III Man . III Man consists of two functional subdomains linked by an Ala-Pro-rich hinge peptide; each subdomain contains a phosphorylation site (16). In the phosphotransfer reactions, III Man is phosphorylated at residue His-10 by phospho-HPr. The phosphoryl group is then transferred to His-175, in the second functional domain of III Man (16,31). Finally, transfer of the phosphoryl group to the sugar then requires the two membrane proteins, IIP Man and IIB Man (4,32). The two subdomains of III Man are complementary when separated in vitro by protease digestion, or in vivo by molecular genetic techniques (16). Recently, Stolz et al. (31) concluded from a study of III Man point mutants that His-86 is required for phosphotransfer between His-10 and His-175 and that His-86 can substitute for His-10 provided that it is on the same III Man polypeptide as His-175.
The deduced amino acid sequences of the V. furnissii II Man complex shared identity with the four subunits of B. subtilis Enzyme II Lev (from 25.7% to 26.1% identity) and K. pneumoniae Enzyme II Sor (from 19% to 36.6% identity). But the greatest identity was with E. coli Enzyme II Man (Fig. 6) Our data therefore suggest that the single gene, manX, which encodes III Man in E. coli, is a fusion product of two genes, manX and manW. Alternatively, the reverse could have occurred, separating one gene into two genes in V. furnissii. This separation also occurs in the B. subtilis lev (27) and K. pneumoniae sor (29) equivalents of E. coli manX. However, in lev and sor, the genes encoding each of the III-like protein domains are adjacent to one another, unlike the separation observed in the V. furnissii man complex.
The DNA fragment p3H1S contains two open reading frames besides manXYZW, but neither is essential for Man phosphorylation by the PTS. One open reading frame, designated manD, encodes a protein with identity to the E. coli enzyme, GlcNAc-6-P deacetylase (33). The enzymes required for GlcNAc catabolism in E. coli are encoded by the nag operon; nagA encodes GlcNAc-6-P deacetylase. The deacetylase is the first enzyme in the GlcNAc dissimilation pathway after the sugar enters the cell as GlcNAc-6-P via the PTS (34). It appears that V. furnissii ManD is functional in E. coli since it complements nagA mutants. The reason for linkage of manD with the man operon is unclear, however, since the V. furnissii nag operon also encodes a GlcNAc-6-P deacetylase (see accompanying article; Ref. 1). The deduced sequences of the two V. furnissii deacetylases are only 29% identical (data not shown). One possibility for the linkage of manD with ptsM in V. furnissii is that the true substrate of the deacetylase is N-acetylmannosamine-6-P. N-Acetylmannosamine is a substrate of E. coli ptsM (35), and linking it with a deacetylase would convert external ManNAc to internal mannosamine-6-P. There are, of course, other deacetylases for derivatives of GlcNAc, such as the NodB proteins and chitin deacetylases. However, the identity/similarity of these sequences to ManD is relatively small (data not shown).
Another open reading frame (manF) linked with manXYZW and manD potentially encodes a protein that is similar to several bacterial aldolases. ManF is not required for mannose uptake. Further analysis, such as transcriptional mapping in wild-type V. furnissii, is required to conclusively demonstrate the co-expression of manXYZW, manD, and manF.
In sum, the V. furnissii ptsM operon has been identified. While it is similar to E. coli ptsM, it exhibits interesting and potentially important differences that may reflect different physiological roles in the two organisms. For example, in E. coli, ptsM is an alternate path for GlcNAc uptake but apparently not in V. furnissii. These results explain our previous observation that a mutation in nagE (which encodes the membrane receptor, II Nag ) prevents V. furnissii from taking up or exhibiting chemotaxis to GlcNAc (9). In view of the complexity of the chitin catabolic cascade in this Vibrio (9,13,36,37), it may be essential that GlcNAc uptake and phosphorylation be limited to a single permease that is maintained under stringent control.